Thermotolerant ribonuclease h

ABSTRACT

Polypeptides having an RNase H activity highly useful in genetic engineering; genes encoding these polypeptides; and a process for genetic engineeringly producing these polypeptides.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a division of application Ser. No.10/380,430, filed Mar. 14, 2003, which is the national stage under 35U.S.C. 371 of PCT/JP01/07930, filed Sep. 13, 2001, which claims priorityfrom JP2000-280785, filed Sep. 14, 2000 and JP2001-064074, filed Mar. 7,2001. The entire contents of prior applications are herein incorporatedby reference.

TECHNICAL FIELD

The present invention relates to a polypeptide, specifically apolypeptide having a ribonuclease H activity which is highly valuablefor genetic engineering. The present invention also relates to a genethat is useful for producing said polypeptide by genetic engineering.The present invention further relates to a method for producing saidpolypeptide by genetic engineering.

BACKGROUND ART

There are endo-type and exo-type ribonucleases (RNA-degrading enzymes).Their substrate specificities are various, and they are involved incomplicated physiological activities. Enzymes such as ribonuclease T₁,ribonuclease T₂, ribonuclease H, ribonuclease P, ribonuclease I,ribonuclease II, ribonuclease III, ribonuclease IV, ribonuclease L areknown to have ribonuclease activities.

Ribonuclease H (hereinafter also referred to as RNase H) was firstisolated from calf thymus by W. H. Stein and P. Hausen in 1969. RNase Hsare currently classified into cellular RNase Hs and viral RNase Hs. Thecellular RNase Hs are widely present in eukaryotes such as variousanimal cells and yeasts and prokaryotes such as Escherichia coli,whereas the viral RNase Hs are present in RNA tumor viruses. Severalkinds of RNase H activities are present in a cell. They require divalentmetal ions such as Mg²⁺ and Mn²⁺.

An RNase H from Escherichia coli is a hydrolase that consists of 155amino acids, has a molecular weight of about 17 kDa and has a substratespecificity of specifically cleaving only the RNA strand in a DNA-RNAhybrid in an endo-type manner. The resulting oligomer has a phosphategroup at the 5′ end and a hydroxyl group at the 3′ end.

RNase HI and RNase HII have been identified as RNase Hs from E. coli. Ithas been shown that RNase HI has the following physiological functionsin the replication of the Col E1 plasmid: 1) it degrades RNAs bound toportions other than the normal replication origin to ensure the normalreplication origin; and 2) it synthesizes an RNA primer specific for thenormal replication origin. The function of RNase HII remains unknown.

It is considered that RNase H increasingly becomes important with thedevelopment of genetic engineering. However, the expression level ofthis enzyme in E. coli is quite low. Then, production of this enzymeusing recombinant DNA techniques has been attempted. RNase Hs producedusing recombinant DNA techniques are now supplied from BRL, AmershamPharmacia Biotech, Takara Shuzo and the like.

These commercially available recombinant RNase Hs are produced usingEscherichia coli as a host (Kanaya et al., The Journal of BiologicalChemistry, 264:11546-11549 (1989)). A method of producing an RNase Hfrom a thermophile, which is much more stable than RNase H from E coli,using E. coli has been reported (Kanaya et al., Dai 2 Kai NipponTanpakukougakukai Nenkai Program/Abstract (1990) pp. 69; Japanese PatentNo. 2533671). However, the enzymatic activity of the RNase H from athermophile produced using E. coli was lower than that of RNase H fromE. coli.

RHase Hs have uses as exemplified below based on the substratespecificities, and attention is paid to RNase Hs as very valuableenzymes:

1) removal of template mRNA upon cDNA cloning;

2) removal of poly(A) region in mRNA; and

3) fragmentation of RNA.

However, as described above, only thermostable RNase Hs of which theproductivities and the enzymatic activities are lower than those ofRNase H from E. coli are available. Thus, development of a thermostableRNase H of which the productivity and the enzymatic activity areequivalent to or more than those of the RNase H from E. coli has beendesired for expanding the uses of RNase H.

OBJECTS OF INVENTION

The main object of the present invention is to provide a polypeptidehaving an RNase H activity which is highly valuable for geneticengineering, a gene encoding said polypeptide and a method for producingsaid polypeptide by genetic engineering.

SUMMARY OF INVENTION

In view of the circumstances as described above, the present inventorshave studied intensively and conducted screening in order to obtain athermostable RNase H. As a result, the present inventors have found athermostable RNase H polypeptide having a high RNase H activity.Furthermore, the present inventors have found that the productivity ofthe thus obtained thermostable RNase H in production using geneticengineering techniques is high. Thus, the present invention has beencompleted.

The present invention is outlines as follows. The first aspect of thepresent invention relates to a thermostable ribonuclease H polypeptide,i.e., a polypeptide having a thermostable ribonuclease H activity, whichis selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO:9, 17, 23,32, 37, 47, 57 or 59, or a portion thereof;

(b) a polypeptide having an amino acid sequence in which at least oneamino acid residue is deleted, added, inserted or substituted in theamino acid sequence of SEQ ID NO:9, 17, 23, 32, 37, 47, 57 or 59; and

(c) a polypeptide having an amino acid sequence that shares at least 71%homology with the amino acid sequence of SEQ ID NO:9, 17, 23, 32, 37,47, 57 or 59.

The second aspect of the present invention relates to a nucleic acidencoding a thermostable ribonuclease H, i.e., a nucleic acid encoding apolypeptide having a thermostable ribonuclease H activity, which isselected from the group consisting of:

(a) a nucleic acid encoding a polypeptide having the amino acid sequenceof SEQ ID NO:9, 17, 23, 32, 37, 47, 57 or 59, or a portion thereof;

(b) a nucleic acid encoding a polypeptide having an amino acid sequencein which at least one amino acid residue is deleted, added, inserted orsubstituted in the amino acid sequence of SEQ ID NO:9, 17, 23, 32, 37,47, 57 or 59;

(c) a nucleic acid having the nucleotide sequence of SEQ ID NO:8, 16,22, 31, 36, 46, 56 or 58;

(d) a nucleic acid in which at least one nucleotide is deleted, added,inserted or substituted in the nucleotide sequence of SEQ ID NO:8, 16,22, 31, 36, 46, 56 or 58 such that the deletion, addition, insertion orsubstitution of the nucleotide results in translation into an amino acidsequence;

(e) a nucleic acid that is hybridizable to any one of the nucleic acidsof (a) to (d) or complementary strands thereof under stringentconditions; and

(f) a nucleic acid having a nucleotide sequence that shares at least 69%homology with the nucleotide sequence of SEQ ID NO:8, 16, 22, 31, 36,46, 56 or 58.

The third aspect of the present invention relates to a recombinant DNAcomprising the nucleic acid of the second aspect.

The fourth aspect of the present invention relates to a transformanttransformed with the recombinant DNA of the third aspect.

The fifth aspect of the present invention relates to a method forproducing a polypeptide having a thermostable ribonuclease H activity,the method comprising:

culturing the transformant of the fourth aspect; and

collecting a polypeptide having a thermostable ribonuclease H activityfrom the culture.

The sixth aspect of the present invention relates to a polypeptidehaving a thermostable ribonuclease H activity, which is obtainable byculturing a transformant into which any one of the plasmids pRHB11,pBCA3Nd2, pPFU220, pTM-RNH, pPHO238, pAFU204, pTLI204 and pTCE207 istransferred. Escherichia coli strains harboring these plasmids aredeposited under Budapest Treaty at International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology under accession numbers FERM BP-7655, FERM BP-7653, FERMBP-7654, FERM BP-7652, FERM BP-7692, FERM BP-7691, FERM BP-7693 and FERMBP-7694, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail.

As used herein, an RNase H refers to a hydrolase that has a substratespecificity of specifically cleaving only the RNA strand in a DNA-RNAhybrid in an endo-type manner, wherein the resulting oligomer has aphosphate group at the 5′ end and a hydroxyl group at the 3′ end.

Although it is not intended to limit the present invention, having athermostable RNase H activity as used herein with respect to apolypeptide means that the polypeptide has an RNase H activity afterincubating it at a temperature of 60° C. or above for 15 minutes.

For example, a thermostable RNase H activity can be determined asfollows.

1 mg of poly(rA) or poly(dT) (both from Amersham Pharmacia Biotech) isdissolved in 1 ml of 40 mM tris-HCl (pH 7.7) containing 1 mM EDTA toprepare a poly(rA) solution and a poly(dT) solution.

The poly(rA) solution (to a final concentration of 20 μg/ml) and thepoly(dT) solution (to a final concentration of 30 μg/ml) are then addedto 40 mM tris-HCl (pH 7.7) containing 4 mM MgCl₂, 1 mM DTT, 0.003% BSAand 4% glycerol. The mixture is reacted at 37° C. for 10 minutes andthen cooled to 4° C. at prepare a poly(rA)-poly(dT) solution.

1 μl of an enzyme solution is added to 100 μl of the poly(rA)-poly(dT)solution. The mixture is reacted at 40° C. for 10 minutes. 10 μl of 0.5M EDTA is added thereto to terminate the reaction. Absorbance at 260 nmis then measured. As a control, 10 μl of 0.5 M EDTA is added to thereaction mixture, the resulting mixture is reacted at 40° C. for 10minutes, and the absorbance is then measured. A value (difference inabsorbance) is obtained by subtracting the absorbance for the controlfrom the absorbance for the reaction in the absence of EDTA. Thus, theconcentration of nucleotide released from poly(rA)-poly(dT) hybrid bythe enzymatic reaction is determined on the basis of the difference inabsorbance. Thus, the thermostable RNase H activity according to thepresent invention can be determined.

Alternatively, the thermostable RNase H activity according to thepresent invention can be determined as follows. 100 μl of a reactionmixture [20 mM HEPES-potassium hydroxide (pH 8.5), 0.01% bovine serumalbumin (Takara Shuzo), 1% dimethyl sulfoxide, 4 mM magnesium acetate,20 μg/ml poly(dT) (Amersham Pharmacia Biotech), 30 μg/ml poly(rA)(Amersham Pharmacia Biotech)] which has been incubated at 40° C. isadded to 1 μl of an enzyme solution of which the activity is to bedetermined. The mixture is reacted at 40° C. for 10 minutes. Thereaction is then terminated by adding 10 μl of 0.5 M EDTA (pH 8.0).Absorbance at 260 nm is then measured.

One unit of an RNase H is defined as an amount of enzyme that increasesA₂₆₀ corresponding to release of 1 nmol of ribonucleotide in 10 minutescalculated according to the following equation:

Unit=[Difference in Absorbance×Reaction Volume (ml)]/0.0152

The polypeptides of the present invention include a polypeptide havingan amino acid sequence in which at least one amino acid residue isdeleted, added, inserted or substituted in the amino acid sequence ofSEQ ID NO:9, 17, 23, 32, 37, 47, 57 or 59 as long as it exhibits athermostable RNase H activity.

A mutation such as deletion, insertion, addition or substitution of anamino acid in an amino acid sequence may be generated in a naturallyoccurring polypeptide. Such mutation may be generated due to apolymorphism or a mutation of the DNA encoding the polypeptide, or dueto a modification of the polypeptide in vivo or during purificationafter synthesis. However, it is known that such a mutated polypeptidemay exhibit a physiological or biological activity substantiallyequivalent to that of a polypeptide without a mutation if such amutation is present in a portion that is not important for the retentionof the activity or the structure of the polypeptide.

This is applicable to a polypeptide in which such a mutation isartificially introduced into an amino acid sequence of a polypeptide. Inthis case, it is possible to generate more various mutations. Forexample, it is known that a polypeptide in which a cysteine residue inthe amino acid sequence of human interleukin-2 (IL-2) is replaced by aserine retains the interleukin-2 activity (Science, 224:1431 (1984)).

Furthermore, it is known that certain polypeptides have peptide regionsthat are not indispensable to their activities. Such peptide regions areexemplified by a signal peptide in a polypeptide to be secretedextracellularly, or a prosequence or pre-prosequence found in aprecursor of a protease. Most of such regions are removed aftertranslation or upon conversion into an active polypeptide. Such apolypeptide has a primary structure different from that of a polypeptidewithout the region to be removed, but finally exhibits an equivalentfunction.

Genes having nucleotide sequences of SEQ ID NOS:8, 16, 22, 31, 36, 46,56 or 58 which are isolated according to the present invention encodepolypeptides having the amino acid sequences of SEQ ID NOS:9, 17, 23,32, 37, 47, 57 or 59, respectively. These polypeptides have thermostableRNase H activities. The polypeptides of the present invention includepolypeptides from which peptide regions that are not indispensable totheir activities have been deleted therefrom.

When a polypeptide is produced by genetic engineering, a peptide chainthat is irrelevant to the activity of the polypeptide of interest may beadded at the amino terminus or the carboxyl terminus of the polypeptide.For example, a fusion polypeptide, in which a portion of an aminoterminus region of a polypeptide that is expressed at a high level inthe host to be used is added at the amino terminus of the polypeptide ofinterest, may be prepared in order to increase the expression level ofthe polypeptide of interest. In another case, a peptide having anaffinity for a specific substance may be added at the amino terminus orthe carboxyl terminus of the polypeptide of interest in order tofacilitate the purification of the expressed polypeptide. The addedpeptide may remain added if it does not have a harmful influence on theactivity of the polypeptide of interest. If necessary, it may beengineered such that it can be removed from the polypeptide of interestby appropriate treatment, for example, by limited digestion with aprotease.

Thus, a polypeptide having an amino acid sequence in which at least oneamino acid residue is deleted, inserted, added or substituted in theamino acid sequence of SEQ ID NO:9, 17, 23, 32, 37, 47, 57 or 59disclosed herein is encompassed by the present invention if it has athermostable RNase H activity.

Furthermore, a polypeptide having an amino acid sequence that shares atleast 71%, preferably 80%, more preferably 90% homology with the aminoacid sequence of SEQ ID NO:9, 17, 23, 32, 37, 47, 57 or 59 disclosedherein is encompassed by the present invention if it has a thermostableRNase H activity.

The homology can be determined using, for example, a computer programDNASIS-Mac (Takara Shuzo), a computer algorithm FASTA (version 3.0;Pearson, W. R. et al., Pro. Natl. Acad. Sci., 85:2444-2448, 1988) or acomputer algorithm BLAST (version 2.0, Altschul et al., Nucleic AcidsRes. 25:3389-3402, 1997).

For example, a polypeptide that shares at least 44% homology with theamino acid sequence of the ribonuclease HII from Bacillus caldotenax(SEQ ID NO:9), at least 47% homology with the amino acid sequence of theribonuclease HIII from Bacillus caldotenax (SEQ ID NO:17), at least 69%homology with the amino acid sequence of the ribonuclease H fromPyrococcus furiosus (SEQ ID NO:23), at least 53% homology with the aminoacid sequence of the ribonuclease H from Thermotoga maritima (SEQ IDNO:59), at least 51% homology with the amino acid sequence of theribonuclease H from Archaeoglobus fulgidus (SEQ ID NO:37), at least 65%homology with the amino acid sequence of the ribonuclease H fromThermococcus litoralis (SEQ ID NO:47), at least 71% homology with theamino acid sequence of the ribonuclease H from Thermococcus celer (SEQID NO:57), or at least 71% homology with the amino acid sequence of theribonuclease H from Pyrococcus horikoshii (SEQ ID NO:32) is encompassedby the present invention if it has a thermostable RNase H activity.

The polypeptide of the present invention can be produced, for example,by (1) purification from a culture of a microorganism producing thepolypeptide of the present invention, or (2) purification from a cultureof a transformant containing a nucleic acid encoding the polypeptide ofthe present invention.

(1) Purification from Culture of Microorganism Producing the Polypeptideof the Present Invention

The microorganism producing the polypeptide of the present invention isexemplified by Bacillus caldotenax (DSM406), Pyrococcus furiosus(DSM3638), Thermotoga maritima (DSM3109), Archaeoglobus fulgidus(DSM4139), Thermococcus litoralis (DSM5473) or Thermococcus celer(DSM2476) which can be purchased from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, or Pyrococcus horikoshii(JCM9974) which can be purchased from the Institute of Physical andChemical Research (RIKEN). The microorganism is cultured underconditions suitable for the growth of the microorganism. Preferably,culture conditions that increase the expression level of the polypeptideof interest are used. The polypeptide of interest produced in the cellsor the culture medium can be purified according to a methodconventionally used for purifying a protein.

A method conventionally used for culturing a thermostable bacterium canbe utilized for the cultivation of the above-mentioned strain. Nutrientsthat can be utilized by the strain are added to the culture medium. Forexample, starch can be used as a carbon source, and Tryptone, peptoneand yeast extract can be used as nitrogen sources. A metal salt such asa magnesium salt, a sodium salt or an iron salt may be added to aculture medium as a trace element. In addition, it may be advantageousto use artificial seawater for the preparation of a culture medium incase of a thermostable marine bacterium, for example.

The culture may be a standing culture or a spinner culture. For example,a dialysis culture method as described in Applied and EnvironmentalMicrobiology, 55:2086-2088 (1992) may be used. It is preferable todetermine the culture conditions and the cultivation time depending onthe strain or the composition of the culture medium to be used such thatthe productivity of the polypeptide becomes maximal.

A cell-free extract is first prepared in order to obtain a polypeptide.The cell-free extract can be prepared, for example, by collecting cellsfrom a culture by centrifugation, filtration or the like and thendisrupting the cells. A cell disruption method highly effective forextracting the enzyme of interest may be selected from sonication,disruption using beads, treatment with a lytic enzyme and the like. Ifthe polypeptide is secreted into a culture supernatant, the polypeptidein the culture supernatant is concentrated by ammonium sulfateprecipitation, ultrafiltration or the like. The concentrated polypeptideis used as a cell-free extract. A method conventionally used forpurifying a protein can be used to isolate the polypeptide from the thusobtained cell-free extract. For example, ammonium sulfate precipitation,ion exchange chromatography, hydrophobic chromatography, gel filtrationchromatography and the like can be used in combination.

(2) Purification from Culture of Transformant Transformed withRecombinant DNA Containing Nucleic Acid Encoding the Polypeptide of thePresent Invention

The polypeptide of the present invention can be obtained from atransformant transformed with a recombinant DNA that contains a nucleicacid encoding the polypeptide of the present invention, for example, anucleic acid having a nucleotide sequence of SEQ ID NO:8, 16, 22, 31,36, 46, 56 or 58. A polypeptide having an amino acid sequence of SEQ IDNO:9 is produced using a nucleic acid having a nucleotide sequence ofSEQ ID NO:8. A polypeptide having an amino acid sequence of SEQ ID NO:17is produced using a nucleic acid having a nucleotide sequence of SEQ IDNO:16. A polypeptide having an amino acid sequence of SEQ ID NO:23 isproduced using a nucleic acid having a nucleotide sequence of SEQ IDNO:22. A polypeptide having an amino acid sequence of SEQ ID NO:32 isproduced using a nucleic acid having a nucleotide sequence of SEQ IDNO:31. A polypeptide having an amino acid sequence of SEQ ID NO:37 isproduced using a nucleic acid having a nucleotide sequence of SEQ IDNO:36. A polypeptide having an amino acid sequence of SEQ ID NO:47 isproduced using a nucleic acid having a nucleotide sequence of SEQ IDNO:46. A polypeptide having an amino acid sequence of SEQ ID NO:57 isproduced using a nucleic acid having a nucleotide sequence of SEQ IDNO:56. A polypeptide having an amino acid sequence of SEQ ID NO:59 isproduced using a nucleic acid having a nucleotide sequence of SEQ IDNO:58.

The polypeptide of the present invention may be purified from a cultureobtained by culturing a transformant into which any one of the plasmidsof the present invention pRHB11, pBCA3Nd2, pPFU220, pTM-RNH, pPHO238,pAFU204, pTLI204 and pTCE207 is transferred.

The host to be transformed is not limited to specific one andexemplified by those conventionally used in a field of recombinant DNAincluding Escherichia coli, Bacillus subtilis, yeast, filamentous fungi,plants, animals, cultured plant cells and cultured animal cells.

For example, the polypeptide of the present invention can be obtainedusing Escherichia coli harboring a plasmid in which the nucleic acid ofthe present invention is linked downstream from a lac promoter or a T7phage promoter under conventional culture conditions, for example, in LBmedium (10 g/l Tryptone, 5 g/l yeast extract, 5 g/l NaCl, pH 7.2)containing 100 μg/ml of ampicillin at 37° C. until logarithmic growthphase, adding isopropyl-β-D-thiogalactopyranoside at a finalconcentration of 1 mM thereto and further culturing at 37° C. to expressthe polypeptide in the cultured cells.

Cells are collected by centrifugation after cultivation, disrupted bysonication, and a supernatant collected by centrifugation is used as acell-free extract. This cell-free extract exhibits a thermostable RNaseH activity. The polypeptide of the present invention can be purifiedfrom the cell-free extract by using known methods such as ion exchangechromatography, gel filtration, hydrophobic chromatography and ammoniumsulfate precipitation. Naturally, a partially purified product obtainedduring the purification process as described above also exhibits anRNase H activity. Since the polypeptide of the present inventionexpressed in Escherichia coli harboring a plasmid linked to the nucleicacid of the present invention is thermostable, the cultured cells and/orthe cell-free extract may be heated, for example, at a temperature of40° C. or above for about 10 minutes to remove heat-denatured insolubleproteins derived from the host in order to purify the polypeptide. Anoptimal temperature or time may be suitably selected for the heattreatment.

As described above, when the polypeptide of the present invention isexpressed at normal temperature (e.g., 37° C.) using a transformantharboring a nucleic acid encoding the polypeptide, the resultingexpression product retains the activity, the thermostability and thelike. That is, the polypeptide of the present invention can assume itsinherent higher-order structure even if it is expressed at a temperaturequite different from the growth temperature of the original producercell.

The nucleic acid of the present invention is a nucleic acid that encodesthe polypeptide of the present invention. Specifically, it is (1) anucleic acid that encodes a polypeptide having the amino acid sequenceof SEQ ID NO:9, 17, 23, 32, 37, 47, 57 or 59, or an amino acid sequencein which at least one amino acid residue is deleted, added, inserted orsubstituted in the sequence and having a thermostable RNase H activity;(2) a nucleic acid having the nucleotide sequence of SEQ ID NO:8, 16,22, 31, 36, 46, 56 or 58; (3) a nucleic acid that has a nucleotidesequence that is hybridizable to the nucleotide sequence of (1) or (2)above under stringent conditions, or that shares at least 69%,preferably 80%, more preferably 90% homology with the nucleotidesequence of (1) or (2) above, and that encodes a polypeptide having athermostable RNase H activity, or the like.

The homology of the nucleotide sequence can be determined using acomputer program DNASIS-Mac, or a computer algorithm FASTA (version 3.0)or BLAST (version 2.0).

As used herein, a nucleic acid means a single-stranded ordouble-stranded DNA or RNA. If the nucleic acid of (2) above is an RNA,it is represented by a nucleotide sequence in which T is replaced by Uin the nucleotide sequence of SEQ ID NO:8, for example.

For example, the nucleic acid of the present invention can be obtainedas follows.

The nucleic acid of (2) above having the nucleotide sequence of SEQ IDNO:8, 16, 22, 31, 36, 46, 56 or 58 can be isolated as follows. A genomicDNA is prepared according to a conventional method from Bacilluscaldotenax (DSM406), Pyrococcus furiosus (DSM3638), Thermotoga maritima(DSM3109), Archaeoglobus fulgidus (DSM4139), Thermococcus litoralis(DSM5473), Thermococcus celer (DSM2476) or Pyrococcus horikoshii(JCM9974) cultured as described above for the polypeptide of the presentinvention. The genomic DNA is used to construct a DNA library. Thenucleic acid can be isolated from the DNA library. Also, the nucleicacid can be obtained by amplifying a nucleic acid having a nucleotidesequence of SEQ ID NO:8, 16, 22, 31, 36, 46, 56 or 58 by a polymerasechain reaction (PCR) using the genomic DNA as a template.

Furthermore, a nucleic acid encoding a polypeptide having a thermostableRNase H activity similar to that of the polypeptide of the presentinvention can be obtained on the basis of the nucleotide sequence of thenucleic acid encoding the polypeptide of the present invention providedby the present invention (e.g., the nucleotide sequence of SEQ ID NO:8,16, 22, 31, 36, 46, 56 or 58). Specifically, a DNA encoding apolypeptide having a thermostable RNase H activity can be screened byusing the nucleic acid encoding the polypeptide of the present inventionor a portion of the nucleotide sequence as a probe for hybridizationfrom a DNA extracted from cells or PCR products obtained using the DNAas a template. Alternatively, a DNA encoding a polypeptide having athermostable RNase H activity can be amplified using a geneamplification method such as a PCR using a primer designed based on theabove-mentioned nucleotide sequence. Additionally, a DNA encoding apolypeptide having a thermostable RNase H activity can be chemicallysynthesized. The nucleic acids of (1) or (3) above can be obtainedaccording to such a method.

A nucleic acid fragment containing only a portion of the nucleic acid ofinterest may be obtained according to the above-mentioned method. Inthis case, the entire nucleic acid of interest can be obtained asfollows. The nucleotide sequence of the obtained nucleic acid fragmentis determined to confirm that the fragment is a portion of the nucleicacid of interest. Hybridization is carried out using the nucleic acidfragment or a portion thereof as a probe. Alternatively, a PCR iscarried out using a primer synthesized on the basis of the nucleotidesequence of the nucleic acid fragment.

“Hybridize under stringent conditions” refers to being capable ofhybridizing under conditions as described in T. Maniatis et al. (eds.),Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring HarborLaboratory (1989), for example, under the following conditions. Amembrane onto which a nucleic acid is immobilized is incubated with aprobe in 6×SSC (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0)containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1%polyvinylpyrrolidone, 0.1% Ficoll 400 and 0.01% denatured salmon spermnucleic acid at 50° C. for 12 to 20 hours. After incubation, themembrane is washed in 2×SSC containing 0.5% SDS at 37° C. while changingthe SSC concentration down to 0.1× and the temperature up to 50° C.until the signal from the immobilized nucleic acid can be distinguishedfrom background, and the probe is then detected. The activity of theprotein encoded by the thus obtained novel nucleic acid is determined asdescribed above, thereby confirming whether or not the nucleic acid isthe nucleic acid of interest.

If an oligonucleotide probe is used, “stringent conditions” refer to,for example, incubation at a temperature of [Tm−25° C.] overnight in asolution containing 6×SSC, 0.5% SDS, 5×Denhardt's and 0.01% denaturedsalmon sperm nucleic acid although it is not intended to limit thepresent invention.

Tm of an oligonucleotide probe or primer can be determined, for example,according to the following equation:

Tm=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)

wherein N is the chain length of the oligonucleotide probe or primer; %G+C is the content of guanine and cytosine residues in theoligonucleotide probe or primer.

If the chain length of the oligonucleotide probe or primer is shorterthan 18 bases, Tm can be estimated, for example, as the sum of theproduct of the number of A+T (adenine and thymine) residues multipliedby 2(° C.) and the product of the number of G+C residues multiplied by4(° C.):

[(A+T)×2+(G+C)×4].

According to the present invention, a nucleic acid which is capable ofhybridizing to the nucleic acid encoding the polypeptide of the presentinvention under stringent conditions is encompassed by the presentinvention as long as it encodes a polypeptide having a thermostableRNase H activity even if it does not have the same nucleotide sequenceas that disclosed herein, as described above.

It is known that one to six codon(s) (a combination of three bases),which defines an amino acid in a gene, is assigned for each amino acid.Thus, many nucleic acids can encode one specific amino acid sequencealthough it depends on the amino acid sequence. Nucleic acids are notnecessarily stable in the nature. Generation of a mutation in anucleotide sequence is not unusual. A mutation generated in a nucleicacid may not alter the encoded amino acid sequence (called a silentmutation). In this case, it can be said that a different nucleic acidencoding the same amino acid sequence is generated. Thus, it cannot bedenied that various nucleic acids encoding the same amino acid sequencecan be generated in the course of passage of an organism containing anucleic acid encoding one specific amino acid sequence. Furthermore, itis not difficult to artificially produce various nucleic acids encodingthe same amino acid sequence if one uses various genetic engineeringtechniques.

For example, if a codon used in an original nucleic acid encoding aprotein of interest is one whose codon usage is low in the host to beused for producing the protein by genetic engineering, the expressionlevel of the protein may be low. In this case, the codon is artificiallyconverted into one frequently used in the host without altering theencoded amino acid sequence aiming at elevating the expression level ofthe protein of interest (e.g., JP-B 7-102146). As described above,various nucleic acids encoding one specific amino acid sequence can beartificially prepared, of course. They may also be generated in thenature.

The nucleic acid encoding the polypeptide of the present invention(e.g., a nucleic acid having the nucleotide sequence of SEQ ID NO:7) canbe ligated to an appropriate vector to construct a recombinant DNA. Thevector to be used for the construction of the recombinant DNA is notspecifically limited. For example, plasmid vectors, phage vectors andvirus vectors can be used. A suitable vector for the object of therecombinant DNA is selected.

Furthermore, a transformant can be produced by introducing therecombinant DNA into an appropriate host. The host to be used for theproduction of a transformant is not specifically limited. Microorganismssuch as bacteria, yeasts and filamentous fungi as well as cultured cellsfrom animals, plants, insects and the like can be used. The polypeptideof the present invention can be produced in large quantities byculturing the transformant to produce the polypeptide of the presentinvention in the culture.

EXAMPLES

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Example 1 Preparation of Rnase H from thermophile Bacillus caldotenax

Bacillus caldotenax YT-G (purchased from Deutsche Sammlung vonMikroorganismen; DSM406) was inoculated into 100 ml of a mediumcontaining 0.2% Tryptone (Difco Laboratories) and 1.5% yeast extract(Difco Laboratories) (pH 6.5), cultured at 60° C. for 140 minutes withshaking and used as a pre-culture. 30 ml of the pre-culture wasinoculated into 3 L of a medium having the same composition and culturedwith aeration at 2.5 L/minute and stirring at 250 rpm at a temperatureof 60° C. for 5 hours.

The cells were collected by centrifuging the culture at 5000×g for 15minutes. 402 g (wet weight) of the cells were suspended in 1000 ml of 50mM tris-HCl buffer (pH 7.5) containing 10 mM mercaptoethanol, 0.5 MNaCl, 1 mM EDTA and 20 μM PAPMSF and disrupted using MINI-Lab (APVGAULIN/RANNIE). Cell debris were removed by centrifugation to recover asupernatant.

A polyethylene imine solution was added to the resulting supernatant ata final concentration of 0.1%. After stirring, the mixture was allowedto stand for 1 hour. A supernatant was then recovered by centrifugation.Ammonium sulfate was added to the supernatant to 50% saturation. Aprecipitate obtained by centrifugation was dissolved in 20 mM tris-HClbuffer (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mM EDTA, 50 mMNaCl and 10% glycerol. The solution was dialyzed against the samebuffer. The dialyzed sample was loaded onto a 280-ml DE52 column(Whatman) equilibrated with the same buffer and non-adsorptive fractionswere collected.

The column was further washed with 420 ml of the buffer used for theequilibration and washing fractions were collected. The non-adsorptivefractions and the washing fractions from the DE52 column chromatographywere mixed together and loaded onto a 240-ml P-11 column (Whatman)equilibrated with 20 mM tris-HCl buffer (pH 7.5) containing 10 mMmercaptoethanol, 0.1 mM EDTA, 50 mM NaCl and 10% glycerol. Elution wasthen carried out using the equilibration buffer containing 0 to 0.5 MNaCl.

The resulting active fractions were placed in a dialysis tube. The tubewas placed on solid polyethylene glycol 20000 fordehydration-concentration at 4° C. The enzyme concentrate was thenloaded onto a 300-ml Superdex G-200 column (Amersham Pharmacia Biotech)equilibrated with 25 mM tris-HCl buffer (pH 7.5) containing 5 mMmercaptoethanol, 0.5 mM EDTA, 30 mM NaCl and 10% glycerol. Elution wascarried out using the buffer used for equilibration to obtain activefractions. The active fractions were loaded onto a 15-mlHeparin-Sepharose column (Amersham Pharmacia Biotech) equilibrated with20 mM tris-HCl buffer (pH 7.5) containing 10 mM mercaptoethanol, 0.1 mMEDTA, 50 mM NaCl and 10% glycerol. Elution was carried out using theequilibration buffer containing 0 to 0.5 M NaCl.

The resulting active fractions were loaded onto a 5-ml Hitrap-SP column(Amersham Pharmacia Biotech) equilibrated with 20 mM tris-HCl buffer (pH7.5) containing 10 mM mercaptoethanol, 0.1 mM EDTA, 50 mM NaCl and 10%glycerol. Elution was carried out using the equilibration buffercontaining 0 to 0.5 M NaCl. The resulting active fractions were loadedonto a 300-ml Superdex G-200 column (Amersham Pharmacia Biotech)equilibrated with 25 mM tris-HCl buffer (pH 7.5) containing 5 mMmercaptoethanol, 0.5 mM EDTA, 30 mM NaCl and 10% glycerol again. Theresulting active fractions were used as an RNase H preparation (anenzyme solution).

A thermostable RNase H activity was measured as follows.

1 mg of poly(rA) or poly(dT) (both from Amersham Pharmacia Biotech) wasdissolved in 1 ml of 40 mM tris-HCl (pH 7.7) containing 1 mM EDTA toprepare a poly(rA) solution and a poly(dT) solution.

The poly(rA) solution (to a final concentration of 20 μg/ml) and thepoly(dT) solution (to a final concentration of 30 μg/ml) were then addedto 40 mM tris-HCl (pH 7.7) containing 4 mM MgCl₂, 1 mM DTT, 0.003% BSAand 4% glycerol. The mixture was reacted at 37° C. for 10 minutes andthen cooled to 4° C. at prepare a poly(rA)-poly(dT) solution.

1 μl of an enzyme solution was added to 100 μl of the poly(rA)-poly(dT)solution. The mixture was reacted at 40° C. for 10 minutes. 10 μl of 0.5M EDTA was added thereto to terminate the reaction. Absorbance at 260 nmwas then measured. As a control, 10 μl of 0.5 M EDTA was added to thereaction mixture, the resulting mixture was reacted at 40° C. for 10minutes, and the absorbance was then measured. A value (difference inabsorbance) was obtained by subtracting the absorbance for the controlfrom the absorbance for the reaction in the absence of EDTA. Thus, theconcentration of nucleotide released from poly(rA)-poly(dT) hybrid bythe enzymatic reaction was determined on the basis of the difference inabsorbance. One unit of an RNase H was defined as an amount of enzymethat increases A₂₆₀ corresponding to release of 1 nmol of ribonucleotidein 10 minutes calculated according to the following equation. If adiluted enzyme solution is used, the value obtained using the followingequation was corrected based on the dilution rate:

Unit=[Difference in Absorbance×Reaction Volume (ml)]/0.0152

Example 2 Cloning of Bacillus caldotenax RNase HII Gene

(1) Preparation of Genomic DNA from Bacillus caldotenax

Bacillus caldotenax YT-G (DSM406) was inoculated into 60 ml of LB medium(1% Tryptone, 0.5% yeast extract and 0.5% NaCl, pH 7.2) and cultured at65° C. for 20 hours. After culturing, the culture was centrifuged tocollect cells. The cells were suspended in 2 ml of 25% sucrose and 50 mMtris-HCl (pH 8.0). 0.2 ml of 10 mg/ml lysozyme chloride (Nacalai Tesque)in water was added thereto. The mixture was reacted at 20° C. for 1hour. After reaction, 12 ml of a mixture containing 150 mM NaCl, 1 mMEDTA and 20 mM tris-HCl (pH 8.0), 0.1 ml of 20 mg/ml proteinase K(Takara Shuzo) and 1 ml of a 10% aqueous solution of sodium laurylsulfate were added to the reaction mixture. The mixture was incubated at37° C. for 1 hour.

2.1 ml of 5 M NaCl and 2 ml of a CTAB-NaCl solution [10%cetyltrimethylammonium bromide (Nacalai Tesque) and 0.7 M NaCl] werethen added to the mixture and the resulting mixture was mixed thoroughlyand incubated at 65° C. for 10 minutes. An equal volume of a mixture ofchloroform/isoamyl alcohol (24:1, v/v) was added thereto. The resultingmixture was gently mixed for 10 minutes and then centrifuged for 10minutes at 10,000×g. After centrifugation, an equal volume of a mixtureof phenol saturated with 100 mM tris-HCl (pH 8.0)/chloroform/isoamylalcohol (25:24:1, v/v) was added to the resulting supernatant. Theresulting mixture was gently mixed for 10 minutes and then centrifugedfor 10 minutes at 10,000×g. After centrifugation, a 0.6 volume of2-propanol was added to the resulting supernatant. The resulting fibrousprecipitate was wound using a glass bar, washed with 70% ethanol inwater, air-dried and then dissolved in 0.5 ml of TE buffer to obtain agenomic DNA solution.

(2) Cloning of Middle Portion of RNase HII Gene

Oligonucleotides BsuII-3 and BsuII-6 represented by SEQ ID NOS:1 and 2were synthesized on the basis of Motif I and Motif III, portionsconserved among amino acid sequences of RNase HIIs from variousorganisms (Biochemistry, 38:605-608 (1999)).

A PCR was carried out in a volume of 100 μl using 1 μl of the Bacilluscaldotenax genomic DNA solution as prepared in Example 2-(1) as atemplate, and 100 pmol each of BsuII-3 and BsuII-6 as primers. TaKaRaTaq (Takara Shuzo) was used as a DNA polymerase for the PCR according tothe attached protocol. The PCR was carried out as follows: 50 cycles of94° C. for 30 seconds, 45° C. for 30 seconds and 72° C. for 1 minute.After reaction, the reaction mixture was subjected to phenol treatmentfollowed by ethanol precipitation to purify a DNA. The resulting DNA wasblunt-ended using T4 DNA polymerase (Takara Shuzo) and then subjected toagarose gel electrophoresis to recover an amplified DNA fragment ofabout 0.4 kb from the gel. The about 0.4-kb DNA fragment was ligatedwith pUC119 (Takara Shuzo) digested with SmaI (Takara Shuzo) using T4DNA ligase (Takara Shuzo). The ligation mixture was used to transformEscherichia coli JM109.

The resulting transformants were cultured to obtain a plasmid 21-12 intowhich the about 0.4-kb DNA fragment was inserted.

(3) Cloning of Upstream Portion of RNase HII Gene

The nucleotide sequence of the inserted fragment of about 0.4 kb in theplasmid 21-12 obtained in Example 2-(2) was determined. OligonucleotidesRNII-S1 and RNII-S2 represented by SEQ ID NOS:3 and 4 were synthesizedon the basis of the determined nucleotide sequence.

The Bacillus caldotenax genomic DNA as prepared in Example 2-(1) wasdigested with BamHI (Takara Shuzo) and ligated with a Sau3AI cassette(Takara Shuzo) using T4 DNA ligase. A procedure was carried outaccording to the protocol attached to TaKaRa LA PCR in vitro cloning kit(Takara Shuzo) using the ligation mixture as a template, RNII-S2 as aprimer for a primary PCR and RNII-S1 as a primer for a secondary PCR. ADNA was purified from the solution after the secondary PCR by phenolextraction followed by ethanol precipitation. The DNA was blunt-endedusing T4 DNA polymerase and then subjected to agarose gelelectrophoresis to recover an amplified DNA fragment of about 1.5 kbfrom the gel. The about 1.5-kb DNA fragment was ligated with pUC119digested with SmaI using T4 DNA ligase. The ligation mixture was used totransform Escherichia coli JM109.

The resulting transformants were cultured to obtain a plasmid B25N16into which the about 1.5-kb DNA fragment was inserted.

(4) Cloning of Entire RNase HII Gene

Oligonucleotides RNII-S5 and RNII-S6 represented by SEQ ID NOS:5 and 6were synthesized on the basis of the nucleotide sequence of the insertedfragment of about 0.4 kb in the plasmid 21-12 as determined in Example2-(3).

A PCR was carried out using the plasmid 21-12 as prepared in Example2-(2) as a template, and RNII-S5 and RNII-S6 as primers. TaKaRa Ex Taq(Takara Shuzo) was used as a DNA polymerase for the PCR according to theattached protocol. The PCR was carried out as follows: 25 cycles of 94°C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds.After reaction, the reaction mixture was subjected to agarose gelelectrophoresis. An amplified DNA fragment of about 0.3 kb was recoveredfrom the gel. The about 0.3-kb DNA fragment was labeled with digoxigeninusing DIG High-prime (Roche Diagnostics).

Southern hybridization was carried out for digests of the Bacilluscaldotenax genomic DNA as prepared in Example 2-(1) with HindIII (TakaraShuzo), Sac I (Takara Shuzo), or HindIII and SacI using thedigoxigenin-labeled DNA as a probe.

Hybridization and detection were carried out using DIG LuminescentDetection Kit (Roche Diagnostics) according to the protocol attachedthereto.

As a result, DNA fragments of about 4.5 kb, about 5.8 kb and about 1.3kb were hybridized with the probe for the digests with HindIII, SacI,and HindIII and SacI, respectively.

Based on these results, the Bacillus caldotenax genomic DNA was digestedwith HindIII and subjected to agarose gel electrophoresis to recover DNAfragments of about 4.5 kb from the gel. The resulting DNA fragments weredigested with SacI and subjected to agarose gel electrophoresis torecover DNA fragments of about 1.3 kb from the gel. The resulting DNAfragments were ligated with pUC19 (Takara Shuzo) digested with HindIIIand SacI using T4 DNA ligase. The ligation mixture was used to transformEscherichia coli HB101.

The resulting transformants were replica-plated onto Hybond-N (AmershamPharmacia Biotech). Colony hybridization was then carried out using theabove-mentioned digoxigenin-labeled probe according to a conventionalmethod. A plasmid pRHB1 was prepared from the thus obtained positiveclone.

The nucleotide sequence of the DNA inserted into pRHB1 was thendetermined. Comparison of the amino acid sequence deduced from thenucleotide sequence with the amino acid sequence of the RNase HII fromBacillus subtilis suggested that a region of about 40 bp from theinitiation codon was missing in the DNA in pRHB1. Then, the full-lengthRNase HII gene was constructed as follows.

B25N16 as prepared in Example 2-(3) was digested with HindIII andsubjected to agarose gel electrophoresis to recover a DNA fragment ofabout 160 bp from the gel. The about 160-bp DNA fragment was ligatedwith pRHB1 digested with HindIII using T4 DNA ligase. The ligationmixture was used to transform Escherichia coli HB101. Plasmids wereprepared from the resulting transformants.

Next, an oligonucleotide RNII-Nde represented by SEQ ID NO:7 wassynthesized on the basis of the presumed nucleotide sequence around theinitiation codon. PCRs were carried out using the plasmids prepared fromthe transformants as templates, and RNII-Nde and RNII-S6 as primers. Aplasmid from which a DNA fragment of about 0.7 kb was amplified wasselected and designated as pRHB11.

The nucleotide sequence of the DNA fragment inserted into the thusobtained plasmid pRHB11 was determined. Analysis of the results revealedan open reading frame (ORF) presumably encoding RNase HII. Thenucleotide sequence of this open reading frame is shown in SEQ ID NO:8.The amino acid sequence of RNase HII deduced from the nucleotidesequence is shown in SEQ ID NO:9.

Escherichia coli HB101 transformed with the plasmid pRHB11 is designatedand indicated as Escherichia coli HB101/pRHB11, and deposited on Sep. 5,2000 (date of original deposit) at International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki 305-8566, Japan under accession number FERM BP-7655.

(5) Expression of Bacillus caldotenax RNase HII Gene

Escherichia coli HB101 transformed with pRHB11 or pRHB1 was inoculatedinto 5 ml of LB medium containing 100 μg/ml of ampicillin and culturedwith shaking at 37° C. overnight. After cultivation, cells collected bycentrifugation were suspended in 0.5 ml of TE buffer and sonicated. Asupernatant obtained by centrifugation was used as a crude cell extract.

10 mM tris-HCl (pH 8.0), 1 mM DTT (Nacalai Tesque), 0.003% BSA (fractionV, Sigma), 4% glycerol, 20 μg/ml poly(dT) (Amersham Pharmacia Biotech)and 30 μg/ml poly(rA) (Amersham Pharmacia Biotech) were mixed together.The mixture was incubated at 37° C. for 10 minutes and used as asubstrate solution for measuring an RNase H activity.

1 μl of 1 M MnCl₂ was added to 100 μl of the substrate solution. Themixture was incubated at 40° C. 10 μl of a 10-fold dilution of the crudecell extract was added to the mixture to initiate a reaction. Afterreacting at 40° C. for 30 minutes, 10 μl of 0.5 M EDTA was added theretoto terminate the reaction. Absorbance at 260 nm was then measured.

As a result, the absorbance at 260 nm from a reaction in which a crudecell extract prepared from Escherichia coli HB101 harboring pRHB11 wasused was clearly higher than that from a reaction in which a crude cellextract prepared from Escherichia coli HB101 harboring pRHB1 was used.Thus, it was demonstrated that pRHB11 contained an RNase HII gene andthat Escherichia coli harboring pRHB11 expressed an RNase H activity.

(6) Preparation of Purified RNase HII Preparation

Escherichia coli HB101 transformed with pRHB11 obtained in Example 2-(4)was inoculated into 1 L of LB medium containing 100 μg/ml of ampicillinand cultured with shaking at 37° C. for 16 hours. After cultivation,cells collected by centrifugation were suspended in 52.3 ml of asonication buffer [50 mM tris-HCl (pH 8.0), 2 mM 2-mercaptoethanol, 10%glycerol, 2 mM phenylmethanesulfonyl fluoride] and sonicated. Asupernatant obtained by centrifuging the sonicated suspension at 12,000rpm for 10 minutes was heated at 60° C. for 15 minutes. It was thencentrifuged again at 12,000 rpm for 10 minutes to collect a supernatant.Thus, 50.0 ml of a heated supernatant was obtained.

The solution was subjected to RESOURSE Q column (Amersham PharmaciaBiotech) equilibrated with Buffer C [50 mM tris-HCl (pH 8.0), 2 mM2-mercaptoethanol, 10% glycerol] and chromatographed using FPLC system(Amersham Pharmacia Biotech). As a result, RNase HII flowed through theRESOURSE Q column.

51 ml of the flow-through RNase HII fraction was subjected to RESOURSE Scolumn (Amersham Pharmacia Biotech) equilibrated with Buffer C andeluted with a linear gradient of 0 to 500 mM NaCl using FPLC system. Afraction containing RNase HII eluted with about 240 mM NaCl wasobtained.

3.0 ml of the RNase HII fraction was subjected in two portions to PD-10column (Amersham Pharmacia Biotech) equilibrated with Buffer Ccontaining 50 mM NaCl. 7.0 ml the resulting eluate was subjected toHiTrap-heparin column (Amersham Pharmacia Biotech) equilibrated withBuffer C containing 50 mM NaCl and eluted with a linear gradient of 50to 550 mM NaCl using FPLC system. A fraction containing RNase HII elutedwith about 310 mM NaCl was obtained.

4.4 ml of the RNase HII fraction was concentrated by ultrafiltrationusing Centricon-10 (Amicon). 280 μl of the concentrate was subjected toSuperdex 200 gel filtration column (Amersham Pharmacia Biotech)equilibrated with 50 mM tris-HCl (pH 8.0) containing 100 mM NaCl and 0.1mM EDTA and eluted with the same buffer. As a result, RNase HII waseluted at a position corresponding to a molecular weight of 35kilodalton. This molecular weight corresponds to that of the RNase HIIin the monomeric form.

The thus eluted RNase HII was used as Bca RNase HII preparation.

The enzymatic activity of the thus obtained Bca RNase HII preparationwas measured as follows.

100 μl of a reaction mixture [20 mM HEPES-potassium hydroxide (pH 7.8),0.01% bovine serum albumin (Takara Shuzo), 1% dimethyl sulfoxide, 10 mMmanganese chloride, 20 μg/ml poly(dT) (Amersham Pharmacia Biotech), 30μg/ml poly(rA) (Amersham Pharmacia Biotech)] which had been incubated at40° C. was added to 1 μl of the Bca RNase HII preparation. The mixturewas reacted at 40° C. for 10 minutes. The reaction was then terminatedby adding 10 μl of 0.5 M EDTA (pH 8.0). Absorbance at 260 nm was thenmeasured.

As a result, an RNase H activity was observed for the Bca RNase HIIpreparation.

Example 3 Cloning of Bacillus caldotenax RNase HIII Gene

(1) Cloning of Fragment of RNase HIII Gene

Primers BsuIII-1, BsuIII-3, BsuIII-6 and BsuIII-8 represented by SEQ IDNOS:10 to 13 for screening a gene encoding RNase HIII were synthesizedbased on the amino acid sequences of regions well conserved amongBacillus subtilis and other organisms determined on the basis of thehomology among the amino acid sequences of RNase HIIIs from Bacillussubtilis [Otani, N. et al., Biochemistry, 38:605-608 (1999)] and otherorganisms.

A first PCR was carried out in a volume of 50 μl using 200 ng of theBacillus caldotenax genomic DNA as prepared in Example 2-(1) as atemplate, and 100 pmol each of BsuIII-1 and BsuIII-8 as primers. Asecond PCR was then carried out in a volume of 100 μl using 1 μl of thereaction mixture as a template, and 100 pmol each of BsuIII-3 andBsuIII-6 as primers. TaKaRa Taq polymerase (Takara Shuzo) was used as aDNA polymerase for the two PCRs according to the attached protocol. ThePCRs were carried out as follows: 25 (the first PCR) or 30 (the secondPCR) cycles of 94° C. for 30 seconds, 45° C. for 30 seconds and 72° C.for 1 minute.

An amplified DNA fragment of about 450 bp was blunt-ended using T4 DNApolymerase (Takara Shuzo) and then subjected to agarose gelelectrophoresis to recover the amplified DNA fragment of about 450 bp.The about 450-bp DNA fragment was ligated with pUC119 (Takara Shuzo)digested with SmaI (Takara Shuzo) using T4 DNA ligase (Takara Shuzo).The ligation mixture was used to transform Escherichia coli JM109.

The resulting transformants were cultured to obtain a plasmid pBCA3204into which the about 450-bp DNA fragment was inserted.

(2) Cloning of RNase HIII Gene Using Southern Hybridization Method

The nucleotide sequence of the DNA fragment inserted in pBCA3204obtained in Example 3-(1) was determined. Primers RNIII-S3 andBcaRNIII-3 represented by SEQ ID NOS:14 and 15 were synthesized on thebasis of the determined nucleotide sequence. A PCR was carried out in avolume of 100 μl using RNIII-S3 and BcaRNIII-3 as primers and pBCA3204as a template. TaKaRa Z-Taq (Takara Shuzo) was used as a DNA polymerasefor the PCR according to the attached protocol. The PCR was carried outas follows: 30 cycles of 98° C. for 0 second, 55° C. for 0 second and72° C. for 20 seconds. After reaction, the reaction mixture wassubjected to phenol-chloroform extraction, ethanol precipitation andagarose gel electrophoresis to recover a DNA fragment of about 0.4 kbfrom the gel. The about 0.4-kb DNA fragment was labeled using DIG DNALabeling Kit (Boehringer Mannheim) to prepare a probe.

20 μg of the Bacillus caldotenax genomic DNA prepared in Example 2-(1)was completely digested with BamHI, EcoRI, HindIII, PstI or XbaI (allfrom Takara Shuzo). The half of each of the digests was then subjectedto agarose gel electrophoresis. The DNAs were transferred from theagarose gel to a nylon membrane using 0.4 N NaOH and fixed at 120° C.for 30 minutes. The membrane was pre-incubated in a sealed bagcontaining 30 ml of a hybridization buffer [43.4 g/L sodium chloride,17.6 g/L sodium citrate, 1% blocking agent (Boehringer Mannheim), 0.1%N-lauroyl sarcosine, 0.02% sodium lauryl sulfate (SDS)] at 60° C. for 4hours and then incubated in a sealed bag containing 5 ml of ahybridization buffer containing the probe at 60° C. for 16 hours.

The membrane was washed twice in 50 ml of 2×SSC (17.5 g/L NaCl, 7.7 g/Lsodium citrate) containing 0.1% SDS at room temperature, and twice in 50ml of 0.5×SSC (4.4 g/L sodium chloride, 1.9 g/L sodium citrate)containing 0.1% SDS at 45° C. Then, an EcoRI fragment of about 8 kb, aPstI fragment of about 4.5 kb and a HindIII fragment of about 1 kb whichhave sequences complementary to the probe were detected using DIGnucleic acid detection kit (Boehringer Mannheim).

The remaining half of the Bacillus caldotenax genomic DNA completelydigested with PstI was subjected to agarose gel electrophoresis. PstIfragments of about 4.5 kb were recovered from the gel. The DNA fragmentswere then ligated with a plasmid vector pTV119N, which had been digestedwith PstI and dephosphorylated with alkaline phosphatase (Takara Shuzo).The ligation mixture was used to transform Escherichia coli JM109.

A PCR was carried out in a volume of 50 μl using RNIII-S3 and BcaRNIII-3as primers, and one of the colonies as a template to select a colonypresumably harboring an RNase HIII gene. TaKaRa-Z Taq (Takara Shuzo) wasused for the PCR according to the attached protocol. The PCR was carriedout as follows: 30 cycles of 98° C. for 0 second, 55° C. for 0 secondand 72° C. for 20 seconds. As a result, it was found that the gene ofinterest was contained in the colony No. 88.

A PCR was carried out using a plasmid prepared from the colony No. 88 asa template, and a primer pair RN-N (Takara Shuzo) and BcaRNIII-3 or aprimer pair M4 (Takara Shuzo) and RNIII-S3 to examine whether or not theentire RNase HIII gene was contained in the plasmid. As a result, it wasanticipated based on the length of the amplification product that theentire RNase HIII gene was contained in the plasmid, which wasdesignated as pBCA3P88.

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRnase HIII Gene

The nucleotide sequence of the DNA fragment inserted into the plasmidpBCA3P88 obtained in Example 3-(2) was determined according to a dideoxymethod.

Analysis of the determined nucleotide sequence revealed the existence ofan open reading frame encoding an amino acid sequence including theN-terminal amino acid sequence of RNase HIII. The nucleotide sequence ofthe open reading frame and the amino acid sequence of RNase HIII deducedfrom the nucleotide sequence are shown in SEQ ID NO:16 and SEQ ID NO:17,respectively.

(4) Construction of Plasmid for Expressing RNase HIII

A PCR was carried out in a volume of 100 μl using the plasmid pBCA3P88as described in Example 3-(2) as a template, BcaRNIIINde represented bySEQ ID NO:18 designed with reference to the sequence around theabove-mentioned open reading frame for RNase HIII and M13 primer M4(Takara Shuzo). Pyrobest DNA polymerase (Takara Shuzo) was used as a DNApolymerase for the PCR according to the attached protocol. The PCR wascarried out as follows: 30 cycles of 94° C. for 30 seconds, 55° C. for30 seconds and 72° C. for 3 minutes. An amplified DNA fragment of about4 kb was digested with NdeI (Takara Shuzo) and subjected to agarose gelelectrophoresis to recover an NdeI fragment of about 1.4 kb from thegel. The about 1.4-kb DNA fragment was ligated with pTV119Nd (a plasmidin which the NcoI site in pTV119N is converted into a NdeI site) whichhad been digested with NdeI and dephosphorylated with alkalinephosphatase (Takara Shuzo). The ligation mixture was used to transformEscherichia coli JM109.

Next, a PCR was carried out in a volume of 50 μl using one of thecolonies as a template, and RV-N (Takara Shuzo) and BcaRNIII-3 (SEQ IDNO:15) as primers in order to screen for a plasmid in which the RNaseHIII gene in the NdeI fragment was linked downstream from the lacpromoter in the vector pTV119Nd. A colony presumably harboring the RNaseHIII gene was then selected. TaKaRa-Z Taq (Takara Shuzo) was used as aDNA polymerase for the PCR according to the attached protocol. The PCRwas carried out as follows: 30 cycles of 98° C. for 0 second, 55° C. for0 second and 72° C. for 20 seconds. As a result, it was found that thecolony No. 2 contained a plasmid in which the RNase HIII gene in theNdeI fragment was linked downstream from the lac promoter in the vectorpTV119Nd. This plasmid was designated as pBCA3Nd2.

The determination of the nucleotide sequence of the DNA fragmentinserted into the plasmid by a dideoxy method revealed that there was nomutation due to the PCR except that the initiation codon GTG was changedto ATG.

Escherichia coli JM109 transformed with the plasmid pBCA3Nd2 isdesignated and indicated as Escherichia coli JM109/pBCA3Nd2, anddeposited on Sep. 5, 2000 (date of original deposit) at InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken 305-8566, Japan under accession number FERMBP-7653.

(5) Preparation of Purified RNase HIII Preparation

Escherichia coli JM109 transformed with pBCA3Nd2 obtained in Example3-(4) was inoculated into 2 L of LB medium containing 100 μg/ml ofampicillin and cultured with shaking at 37° C. for 16 hours. Aftercultivation, cells collected by centrifugation were suspended in 39.6 mlof a sonication buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12,000 rpm for 10 minutes washeated at 60° C. for 15 minutes. It was then centrifuged again at 12,000rpm for 10 minutes to collect a supernatant. Thus, 39.8 ml of a heatedsupernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HIII flowed through the RESOURSE Q column.

45 ml of the flow-through RNase HIII fraction was dialyzed against 2 Lof Buffer B [50 mM tris-HCl (pH 7.0), 1 mM EDTA] for 2 hours. Thedialysis was repeated for two more times under the same conditions. 55.8ml of the dialyzed enzyme solution was subjected to RESOURSE S column(Amersham Pharmacia Biotech) equilibrated with Buffer B and eluted witha linear gradient of 0 to 500 mM NaCl using FPLC system. A fractioncontaining RNase HIII eluted with about 105 mM NaCl was obtained.

Buffer B containing 1 M NaCl was added to 7.0 ml of the fraction to makethe NaCl concentration to 150 mM. The mixture was subjected toHiTrap-heparin column (Amersham Pharmacia Biotech) equilibrated withBuffer B containing 150 mM NaCl. As a result, RNase HIII flowed throughthe HiTrap-heparin column.

7.5 ml of the flow-through RNase HIII fraction was concentrated byultrafiltration using Centricon-10 (Millipore). 190 μl of theconcentrate was subjected to Superdex 200 gel filtration column(Amersham Pharmacia Biotech) equilibrated with 50 mM tris-HCl (pH 7.0)containing 100 mM NaCl and 0.1 mM EDTA and eluted with the same buffer.As a result, RNase HIII was eluted at a position corresponding to amolecular weight of 33 kilodalton. This molecular weight corresponds tothat of the RNase HIII in the monomeric form.

The thus eluted RNase HIII was used as Bca RNase HIII preparation.

The enzymatic activity of the thus obtained Bca RNase HIII preparationwas measured as follows.

100 μl of a reaction mixture [20 mM HEPES-potassium hydroxide (pH 7.8),0.01% bovine serum albumin (Takara Shuzo), 1% dimethyl sulfoxide, 4 mMmagnesium acetate, 20 μg/ml poly(dT) (Amersham Pharmacia Biotech), 30μg/ml poly(rA) (Amersham Pharmacia Biotech)] which had been incubated at40° C. was added to 1 μl of the Bca RNase HIII preparation. The mixturewas reacted at 40° C. for 10 minutes. The reaction was terminated byadding 10 μl of 0.5 M EDTA (pH 8.0). Absorbance at 260 nm was thenmeasured.

As a result, an RNase H activity was observed for the Bca RNase HIIIpreparation.

Example 4 Cloning of Pyrococcus furiosus RNase HII Gene

(1) Preparation of Genomic DNA from Pyrococcus furiosus

2 L of a medium containing 1% Tryptone (Difco Laboratories), 0.5% yeastextract (Difco Laboratories), 1% soluble starch (Nacalai Tesque), 3.5%Jamarine S Solid (Jamarine Laboratory), 0.5% Jamarine S Liquid (JamarineLaboratory), 0.003% MgSO₄, 0.001% NaCl, 0.0001% FeSO₄·7H₂O, 0.0001%COSO₄, 0.0001% CaCl₂·7H₂O, 0.0001% ZnSO₄, 0.1 ppm CuSO₄·5H₂O, 0.1 ppmKAl(SO₄)₂, 0.1 ppm H₃BO₄, 0.1 ppm Na₂MoO₄·2H₂O and 0.25 ppm NiCl₂·6H₂Owas placed in a 2-L medium bottle, sterilized at 120° C. for 20 minutes,bubbled with nitrogen gas to remove dissolved oxygen, then Pyrococcusfuriosus (purchased from Deutsche Sammlung von Mikroorganismen; DSM3638)was inoculated into the medium and cultured at 95° C. for 16 hourswithout shaking. After cultivation, cells were collected bycentrifugation.

The resulting cells were then suspended in 4 ml of 25% sucrose, 50 mMtris-HCl (pH 8.0). 0.4 ml of 10 mg/ml lysozyme chloride (Nacalai Tesque)in water was added thereto. The mixture was reacted at 20° C. for 1hour. After reaction, 24 ml of a mixture containing 150 mM NaCl, 1 mMEDTA and 20 mM tris-HCl (pH 8.0), 0.2 ml of 20 mg/ml proteinase K(Takara Shuzo) and 2 ml of 10% aqueous solution of sodium lauryl sulfatewere added to the reaction mixture. The mixture was incubated at 37° C.for 1 hour.

After reaction, the mixture was subjected to phenol-chloroformextraction followed by ethanol precipitation to prepare about 1 mg ofgenomic DNA.

(2) Cloning of RNase HII Gene

The entire genomic sequence of Pyrococcus horikoshii was published[Kawarabayashi, Y. et al., DNA Research, 5:55-76 (1998)]. The existenceof one gene encoding a homologue of RNase HII (PH1650) in the genome wasknown (SEQ ID NO:19, the home page of National Institute of Technologyand Evaluation: www.nite.go.jp/).

Homology between the PH1650 gene (SEQ ID NO:19) and the partiallypublished genomic sequence of Pyrococcus furiosus (the home page ofUniversity of Utah, Utah Genome Center:www.genome.utah.edu/sequence.html) was searched. As a result, a highlyhomologous sequence was found.

Primers 1650Nde (SEQ ID NO:20) and 1650Bam (SEQ ID NO:21) weresynthesized on the basis of the homologous sequence.

A PCR was carried out in a volume of 100 μl using 200 ng of thePyrococcus furiosus DNA obtained in Example 4-(1) as a template, and 20pmol each of 1650Nde and 1650Bam as primers. TaKaRa Ex Taq (TakaraShuzo) was used as a DNA polymerase for the PCR according to theattached protocol. The PCR was carried out as follows: 30 cycles of 94°C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 1 minute. Anamplified DNA fragment of about 0.7 kb was digested with NdeI and BamHI(both from Takara Shuzo). The resulting DNA fragment was insertedbetween the NdeI site and the BamHI site in a plasmid vector pET3a(Novagen) to make a plasmid pPFU220.

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequence of the DNA fragment inserted into pPFU220obtained in Example 4-(2) was determined according to a dideoxy method.

Analysis of the determined nucleotide sequence revealed the existence ofan open reading frame presumably encoding RNase HII. The nucleotidesequence of the open reading frame is shown in SEQ ID NO:22. The aminoacid sequence of RNase HII deduced from the nucleotide sequence is shownin SEQ ID NO:23.

Escherichia coli JM109 transformed with the plasmid pPFU220 isdesignated and indicated as Escherichia coli JM109/pPFU220, anddeposited on Sep. 5, 2000 (date of original deposit) at InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki 305-8566, Japan under accession number FERMBP-7654.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli HMS174(DE3) (Novagen) was transformed with pPFU220obtained in Example 4-(2). The resulting Escherichia coli HMS174(DE3)harboring pPFU220 was inoculated into 2 L of LB medium containing 100μg/ml of ampicillin and cultured with shaking at 37° C. for 16 hours.After cultivation, cells collected by centrifugation were suspended in66.0 ml of a sonication buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12,000 rpm for 10 minutes washeated at 80° C. for 15 minutes. It was then centrifuged again at 12,000rpm for 10 minutes to collect a supernatant. Thus, 61.5 ml of a heatedsupernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

60.0 ml of the flow-through RNase HII fraction was subjected to RESOURSES column (Amersham Pharmacia Biotech) equilibrated with Buffer A andeluted with a linear gradient of 0 to 500 mM NaCl using FPLC system. Afraction containing RNase HII eluted with about 150 mM NaCl wasobtained.

2.0 ml of the RNase HII fraction was concentrated by ultrafiltrationusing Centricon-10 (Millipore). 250 μl of the concentrate was subjectedto Superdex 200 gel filtration column (Amersham Pharmacia Biotech)equilibrated with 50 mM tris-HCl (pH 8.0) containing 100 mM NaCl and 0.1mM EDTA and eluted with the same buffer. As a result, RNase HII waseluted at a position corresponding to a molecular weight of 17kilodalton. This molecular weight corresponds to that of the RNase HIIin the monomeric form.

The thus eluted RNase HII was used as Pfu RNase HII preparation.

The enzymatic activity of the thus obtained Pfu RNase HII preparationwas measured as described in Example 3-(5). As a result, an RNase Hactivity was observed for the Pfu RNase HII preparation.

Example 5 Cloning of Thermotoga maritima RNase HII Gene

(1) Preparation of Genomic DNA from Thermotoga maritima

2 L of a medium containing 1% Tryptone, 0.5% yeast extract, 1% solublestarch, 3.5% Jamarine S Solid, 0.5% Jamarine S Liquid, 0.003% MgSO₄,0.001% NaCl, 0.0001% FeSO₄·7H₂O, 0.0001% COSO₄, 0.0001% CaCl₂·7H₂O,0.0001% ZnSO₄, 0.1 ppm CuSO₄·5H₂O, 0.1 ppm KAl(SO₄)₂, 0.1 ppm H₃BO₃, 0.1ppm Na₂MoO₄·2H₂O and 0.25 ppm NiCl₂·6H₂O was placed in a 2-L mediumbottle, sterilized at 120° C. for 20 minutes, bubbled with nitrogen gasto remove dissolved oxygen, then Thermotoga maritima (purchased fromDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSM3109)was inoculated into the medium and cultured at 85° C. for 16 hourswithout shaking.

Cells collected by centrifugation from 300 ml of the culture were thensuspended in 3 ml of TE buffer [10 mM tris-HCl (pH 7.5), 1 mM EDTA]. 150μl of 10% aqueous solution of sodium lauryl sulfate (Nacalai Tesque) and15 μl of 20 mg/ml proteinase K (Takara Shuzo) were added thereto. Themixture was incubated at 37° C. for 1 hour.

After reaction, 0.5 ml of 5 M NaCl was added to the mixture. Afterthoroughly mixing, 0.4 ml of a CTAB-NaCl solution [10%cetyltrimethylammonium bromide (Nacalai Tesque), 0.7 M NaCl] was addedto the mixture. After thoroughly mixing, the mixture was incubated at65° C. for 10 minutes. 1.5 ml of a mixture of chloroform/isoamyl alcohol(24:1, v/v) was added thereto. The mixture was gently mixed for 10minutes and centrifuged at 20,000×g for 5 minutes. After centrifugation,an equal volume of a mixture of phenol saturated with 100 mM tris-HCl(pH 8.0)/chloroform/isoamyl alcohol (25:24:1, v/v) was added to theresulting supernatant. The mixture was gently mixed for 10 minutes andthen centrifuged at 20,000×g for 5 minutes. After centrifugation, a 0.6volume of 2-propanol was added to the supernatant. The precipitateobtained by centrifugation at 10,000×g for 5 minutes was washed with 70%ethanol in water, air-dried and then dissolved in 200 μl of TE to obtaina genomic DNA solution.

(2) Cloning of RNase HII Gene

Oligonucleotides 915-F1, 915-F2, 915-R1 and 915-R2 represented by SEQ IDNOS:24 to 27 were synthesized on the basis of the nucleotide sequence ofa portion that had been identified as an RNase HII gene in thenucleotide sequence of the genomic DNA of Thermotoga maritima(www.tigr.org/tdb/CMR/btm/htmls/SplashPage.html) in order to obtain anamplified DNA fragment containing an RNase HII gene by carrying out aPCR using the Thermotoga maritima genomic DNA as a template.

PCRs were carried out using the Thermotoga maritima genomic DNA asprepared in Example 5-(1) as a template, and 915-F1 and 915-R1, 915-F1and 915-R2, 915-F2 and 915-R1, or 915-F2 and 915-R2 as a primer pair.TaKaRa Ex Taq (Takara Shuzo) was used as a DNA polymerase for the PCRsaccording to the attached protocol. The PCRs were carried out asfollows: 25 cycles of 95° C. for 0.5 minute, 55° C. for 0.5 minute and72° C. for 1.5 minute. After reactions, the respective PCR products weresubjected to agarose gel electrophoresis to extract and purify amplifiedDNA fragments of about 0.7 kb.

The DNAs amplified using a primer pair 915-F1 and 915-R1 or 915-F1 and915-R2 were digested with HindIII and XbaI (both from Takara Shuzo) andligated with pUC19 (Takara Shuzo) digested with HindIII and XbaI usingT4 DNA ligase (Takara Shuzo). The ligation mixture was used to transformEscherichia coli JM109.

The resulting transformants were cultured to prepare plasmid DNAs intowhich the about 0.7-kb DNA fragments were inserted. As a result,plasmids No. 1 and No. 2 having DNA fragments amplified using 915-F1 and915-R1, and plasmids No. 3 and No. 4 having DNAs amplified using 915-F1and 915-R2 were obtained.

In addition, the DNAs amplified using a primer pair 915-F2 and 915-R1 or915-F2 and 915-R2 were doubly digested with NcoI (Takara Shuzo) and XbaIand ligated with pTV119N (Takara Shuzo) doubly digested with NcoI andXbaI using T4 DNA ligase. The ligation mixture was used to transformEscherichia coli JM109.

The resulting transformants were cultured to prepare plasmid DNAs intowhich the about 0.7-kb DNA fragments were inserted. As a result,plasmids No. 5 and No. 6 having DNA fragments amplified using 915-F2 and915-R1, and a plasmid No. 7 having a DNA amplified using 915-F2 and915-R2 were obtained.

Escherichia coli JM109 transformed with the plasmid No. 7 is designatedand indicated as Escherichia coli JM109/pTM-RNH, and deposited on Sep.5, 2000 (date of original deposit) at International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki 305-8566, Japan under accession number FERM BP-7652.

(3) Expression of Thermotoga maritima RNase HII Gene

Escherichia coli JM109 transformed with one of the plasmids No. 1 to 7or pUC19 was inoculated into 5 ml of LB medium (10 g/L Tryptone, 5 g/Lyeast extract, 5 g/L NaCl, pH 7.2) containing 100 μg/ml of ampicillinand cultured with shaking at 37° C. When the absorbance at 660 nmreached 0.5, isopropyl-β-D-thiogalactopyranoside was added thereto to afinal concentration of 1 mM and the cells were cultured overnight. Aftercultivation, cells collected by centrifugation were suspended in 1 ml ofTE buffer and sonicated. The sonicated suspension was heated at 80° C.for 10 minute. A supernatant obtained by centrifugation was used as acrude cell extract.

Absorbance was measured using the crude cell extract as described inExample 2-(5).

As a result, when reactions were carried out in the presence of MnCl₂,the absorbance at 260 nm from each of the reactions in which crude cellextracts prepared from Escherichia coli JM109 harboring the plasmid No.3, 5, 6 or 7 were used was clearly higher than that from a reaction inwhich a crude extract prepared from Escherichia coli JM109 harboringpUC19 was used. Thus, it was demonstrated that the plasmids No. 3, 5, 6and 7 contained RNase HII genes and that Escherichia coli harboring oneof these plasmids expressed an RNase H activity.

The nucleotide sequences of the DNA fragments inserted into the plasmidswhich were demonstrated to express RNase H activities in Escherichiacoli were determined. Analysis of the determined nucleotide sequencesrevealed an open reading frame presumably encoding RNase HII. Thenucleotide sequence of the open reading frame is shown in SEQ ID NO:58.The amino acid sequence of RNase HII deduced from the nucleotidesequence is shown in SEQ ID NO:59. Then, it was found that one basesubstitution that was presumably generated upon the PCR was observed inthe nucleotide sequence of the DNA fragment inserted in the plasmid No.7, resulting in the change in the encoded amino acid residue.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli JM109 was transformed with the plasmid No. 7 (pTM-RNH)obtained in Example 5-(2). The resulting Escherichia coli JM109harboring pTM-RNH was inoculated into 1 L of LB medium containing 100μg/ml of ampicillin and cultured with shaking at 37° C. for 16 hours.After cultivation, cells collected by centrifugation were suspended in31.0 ml of a sonication buffer [50 mM tris-HCl (pH 8.0), 2 mM2-mercaptoethanol, 10% glycerol, 2 mM phenylmethanesulfonyl fluoride]and sonicated. A supernatant obtained by centrifuging the sonicatedsuspension at 12,000 rpm for 10 minutes was heated at 70° C. for 15minutes. It was then centrifuged again at 12,000 rpm for 10 minutes tocollect a supernatant. Thus, 32.0 ml of a heated supernatant wasobtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer C [50 mM tris-HCl (pH 8.0),2 mM 2-mercaptoethanol, 10% glycerol] and chromatographed using FPLCsystem (Amersham Pharmacia Biotech). As a result, RNase HII flowedthrough the RESOURSE Q column.

32.5 ml of the flow-through RNase HII fraction was subjected to RESOURSES column (Amersham Pharmacia Biotech) equilibrated with Buffer C andeluted with a linear gradient of 0 to 500 mM NaCl using FPLC system. Afraction containing RNase HII eluted with about 240 mM NaCl wasobtained.

2.0 ml of the RNase HII fraction was subjected to PD-10 column (AmershamPharmacia Biotech) equilibrated with Buffer C containing 50 mM NaCl. 3.5ml of the resulting eluate was subjected to HiTrap-heparin column(Amersham Pharmacia Biotech) equilibrated with Buffer C containing 50 mMNaCl and eluted with a linear gradient of 50 to 550 mM NaCl using FPLCsystem. As a result, a fraction containing RNase HII eluted with about295 mM NaCl was obtained.

The thus eluted RNase HII was used as Tma RNase HII preparation.

The enzymatic activity of the thus obtained Tma RNase HII preparationwas measured as described in Example 2-(6). As a result, an RNase Hactivity was observed for the Tma RNase HII preparation.

Example 6 Cloning of RNase HII Gene from Pyrococcus horikoshii

(1) Preparation of Genomic DNA from Pyrococcus horikoshii

2 L of a medium containing 1% Tryptone (Difco Laboratories), 0.5% yeastextract (Difco Laboratories), 1% soluble starch (Nacalai Tesque), 3.5%Jamarine S Solid (Jamarine Laboratory), 0.5% Jamarine S Liquid (JamarineLaboratory), 0.003% MgSO₄, 0.001% NaCl, 0.0001% FeSO₄·7H₂O, 0.0001%COSO₄, 0.0001% CaCl₂·7H₂O, 0.0001% ZnSO₄, 0.1 ppm CuSO₄·5H₂O, 0.1 ppmKAl(SO₄)₂, 0.1 ppm H₃BO₄, 0.1 ppm Na₂MoO₄·2H₂O and 0.25 ppm NiCl₂·6H₂Owas placed in a 2-L medium bottle, sterilized at 120° C. for 20 minutes,bubbled with nitrogen gas to remove dissolved oxygen, then Pyrococcushorikoshii OT3 (purchased from the Institute of Physical and ChemicalResearch (RIKEN); JCM9974) was inoculated into the medium and culturedat 95° C. for 16 hours without shaking. After cultivation, cells werecollected by centrifugation.

The cells were then suspended in 4 ml of 25% sucrose, 50 mM tris-HCl (pH8.0). 0.4 ml of 10 mg/ml lysozyme chloride (Nacalai Tesque) in water wasadded thereto. The mixture was reacted at 20° C. for 1 hour. Afterreaction, 24 ml of a mixture containing 150 mM NaCl, 1 mM EDTA and 20 mMtris-HCl (pH 8.0), 0.2 ml of 20 mg/ml proteinase K (Takara Shuzo) and 2ml of 10% aqueous solution of sodium lauryl sulfate were added to thereaction mixture. The mixture was incubated at 37° C. for 1 hour.

After reaction, the mixture was subjected to phenol-chloroformextraction followed by ethanol precipitation to prepare about 1 mg ofgenomic DNA.

(2) Cloning of RNase HII Gene

The entire genomic sequence of the Pyrococcus horikoshii has beenpublished [DNA Research, 5:55-76 (1998)]. The existence of one geneencoding a homologue of RNase HII (PH1650) was known (SEQ ID NO:28, thehome page of National Institute of Technology and Evaluation:www.nite.go.jp/).

Primers PhoNde (SEQ ID NO:29) and PhoBam (SEQ ID NO:30) were synthesizedon the basis of the sequence of the PH1650 gene (SEQ ID NO:28).

A PCR was carried out using 100 ng of the Pyrococcus horikoshii DNAprepared in Example 6-(1) as a template, and 20 pmol each of PhoNde andPhoBam as primers in a volume of 100 μl. TaKaRa Ex Taq (Takara Shuzo)was used as a DNA polymerase for the PCR according to the attachedprotocol. The PCR was carried out as follows: 40 cycles of 94° C. for 30seconds, 55° C. for 30 seconds and 72° C. for 1 minute. An amplified DNAfragment of about 0.7 kb was digested with NdeI and BamHI (both fromTakara Shuzo). Then, a plasmid pPHO238 was constructed by incorporatingthe resulting DNA fragment between NdeI and BamHI sites in a plasmidvector pET3a (Novagen).

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequence of the DNA fragment inserted into pPHO238obtained in Example 6-(2) was determined according to a dideoxy method.

Analysis of the determined nucleotide sequence revealed an open readingframe presumably encoding RNase HII. The nucleotide sequence of the openreading frame is shown in SEQ ID NO:31. The amino acid sequence of RNaseHII deduced from the nucleotide sequence is shown in SEQ ID NO:32.

Escherichia coli JM109 transformed with the plasmid pPHO238 isdesignated and indicated as Escherichia coli JM109/pPHO238, anddeposited on Feb. 22, 2001 (date of original deposit) at InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki 305-8566, Japan under accession number FERMBP-7692.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli HMS174(DE3) (Novagen) was transformed with pPHO238obtained in Example 6-(2). The resulting Escherichia coli HMS174(DE3)harboring pPHO238 was inoculated into 1 L of LB medium containing 100μg/ml of ampicillin and cultured with shaking at 37° C. for 16 hours.After cultivation, cells collected by centrifugation were suspended in34.3 ml of a sonication buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12,000 rpm for 10 minutes washeated at 80° C. for 15 minutes. It was then centrifuged again at 12,000rpm for 10 minutes to collect a supernatant. Thus, 33.5 ml of a heatedsupernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

35.0 ml of the flow-through RNase HII fraction was dialyzed against 2 Lof Buffer B (50 mM tris-HCl (pH 7.0), 1 mM EDTA) for 2 hours. Thedialysis was repeated two more times. 34.5 ml of the dialyzed enzymesolution was subjected to RESOURSE S column (Amersham Pharmacia Biotech)equilibrated with Buffer B and eluted with a linear gradient of 0 to 500mM NaCl using FPLC system. A fraction containing RNase HII eluted withabout 155 mM NaCl was obtained.

Buffer B was added to 4.0 ml of the fraction to make the NaClconcentration to 50 mM. The mixture was subjected to HiTrap-heparincolumn (Amersham Pharmacia Biotech) equilibrated with Buffer Bcontaining 50 mM NaCl and eluted with a linear gradient of 50 to 550 mMNaCl using FPLC system. As a result, a fraction containing RNase HIIeluted with about 160 mM NaCl was obtained.

6.9 ml of the RNase HII fraction was concentrated by ultrafiltrationusing Centricon-10 (Millipore). Two portions each separated from 250 μlof the concentrate were subjected to Superose 6 gel filtration column(Amersham Pharmacia Biotech) equilibrated with 50 mM tris-HCl (pH 7.0)containing 100 mM NaCl and 0.1 mM EDTA and eluted with the same buffer.As a result, RNase HII was eluted at a position corresponding to amolecular weight of 24.5 kilodalton. This molecular weight correspondsto that of the RNase HII in the monomeric form.

The RNase HII eluted as described above was used as Pho RNase HIIpreparation.

The enzymatic activity of the thus obtained Pho RNase HII preparationwas measured as described in Example 3-(5). As a result, an RNase Hactivity was observed for the Pho RNase HII preparation.

Example 7 Cloning of RNase HII Gene from Archaeoglobus fulgidus

(1) Preparation of Genomic DNA from Archaeoglobus fulgidus

Cells of Archaeoglobus fulgidus (purchased from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH; DSM4139) collected from 8 ml of aculture was suspended in 100 μl of 25% sucrose, 50 mM tris-HCl (pH 8.0)20 μl of 0.5 M EDTA and 10 μl of 10 mg/ml lysozyme chloride (NacalaiTesque) in water was added thereto. The mixture was reacted at 20° C.for 1 hour. After reaction, 800 μl of a mixture containing 150 mM NaCl,1 mM EDTA and 20 mM tris-HCl (pH 8.0), 10 μl of 20 mg/ml proteinase K(Takara Shuzo) and 50 μl of 10% aqueous solution of sodium laurylsulfate were added to the reaction mixture. The mixture was incubated at37° C. for 1 hour. After reaction, the mixture was subjected tophenol-chloroform extraction, ethanol precipitation and air-drying, andthen dissolved in 50 μl of TE to obtain a genomic DNA solution.

(2) Cloning of RNase HII Gene

The entire genomic sequence of the Archaeoglobus fulgidus has beenpublished [Klenk, H P et al., Nature, 390:364-370 (1997)]. The existenceof one gene encoding a homologue of RNase HII (AF0621) was known (SEQ IDNO:33, www.tigr.org/tdb/CMR/btm/htmls/SplashPage.html).

Primers AfuNde (SEQ ID NO:34) and AfuBam (SEQ ID NO:35) were synthesizedon the basis of the sequence of the AF0621 gene (SEQ ID NO:33).

A PCR was carried out using 30 ng of the Archaeoglobus fulgidus genomicDNA prepared in Example 7-(1) as a template, and 20 pmol each of AfuNdeand AfuBam as primers in a volume of 100 μl. Pyrobest DNA polymerase(Takara Shuzo) was used as a DNA polymerase for the PCR according to theattached protocol. The PCR was carried out as follows: 40 cycles of 94°C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 1 minute. Anamplified DNA fragment of about 0.6 kb was digested with NdeI and BamHI(both from Takara Shuzo). Then, a plasmid pAFU204 was constructed byincorporating the resulting DNA fragment between NdeI and BamHI sites ina plasmid vector pTV119Nd (a plasmid in which the NcoI site in pTV119Nis converted into a NdeI site).

(3) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequence of the DNA fragment inserted into pAFU204obtained in Example 7-(2) was determined according to a dideoxy method.

Analysis of the determined nucleotide sequence revealed an open readingframe presumably encoding RNase HII. The nucleotide sequence of the openreading frame is shown in SEQ ID NO:36. The amino acid sequence of RNaseHII deduced from the nucleotide sequence is shown in SEQ ID NO:37.

Escherichia coli JM109 transformed with the plasmid pAFU204 isdesignated and indicated as Escherichia coli JM109/pAFU204, anddeposited on Feb. 22, 2001 (date of original deposit) at InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki 305-8566, Japan under accession number FERMBP-7691.

(4) Preparation of Purified RNase HII Preparation

Escherichia coli JM109 was transformed with pAFU204 obtained in Example7-(2). The resulting Escherichia coli JM109 harboring pAFU204 wasinoculated into 2 L of LB medium containing 100 μg/ml of ampicillin andcultured with shaking at 37° C. for 16 hours. After cultivation, cellscollected by centrifugation were suspended in 37.1 ml of a sonicationbuffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mM phenylmethanesulfonylfluoride] and sonicated. A supernatant obtained by centrifuging thesonicated suspension at 12,000 rpm for 10 minutes was heated at 70° C.for 15 minutes. It was then centrifuged again at 12,000 rpm for 10minutes to collect a supernatant. Thus, 40.3 ml of a heated supernatantwas obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

The flow-through RNase HII fraction was subjected to RESOURSE S column(Amersham Pharmacia Biotech) equilibrated with Buffer A andchromatographed using FPLC system (Amersham Pharmacia Biotech). As aresult, RNase HII flowed through the RESOURSE S column.

40.0 ml of the flow-through RNase HII fraction was dialyzed against 2 Lof Buffer B (50 mM tris-HCl (pH 7.0), 1 mM EDTA) containing 50 mM NaClfor 2 hours. The dialysis was repeated two more times. 40.2 ml of thedialyzed enzyme solution was subjected to HiTrap-heparin column(Amersham Pharmacia Biotech) equilibrated with Buffer B containing 50 mMNaCl and eluted with a linear gradient of 50 to 550 mM NaCl using FPLCsystem. As a result, a fraction containing RNase HII eluted with about240 mM NaCl was obtained.

7.8 ml of the RNase HII fraction was concentrated by ultrafiltrationusing Centricon-10 (Millipore). Four portions each separated from about600 μl of the concentrate were subjected to Superose 6 gel filtrationcolumn (Amersham Pharmacia Biotech) equilibrated with 50 mM tris-HCl (pH7.0) containing 100 mM NaCl and 0.1 mM EDTA and eluted with the samebuffer. As a result, RNase HII was eluted at a position corresponding toa molecular weight of 30.0 kilodalton. This molecular weight correspondsto that of the RNase HII in the monomeric form.

The RNase HII eluted as described above was used as Afu RNase HIIpreparation.

The enzymatic activity of the thus obtained Afu RNase HII preparationwas measured as described in Example 3-(5). As a result, an RNase Hactivity was observed for the Afu RNase HII preparation.

Example 8 Cloning of Thermococcus litoralis RNase HII Gene

(1) Preparation of Genomic DNA from Thermococcus litoralis

Thermococcus litoralis (purchased from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH; DSM5473) cells were collectedfrom 11 ml of culture. The cells were suspended in 500 μl of 25% sucroseand 50 mM tris-HCl (pH 8.0). 100 μl of 0.5 M EDTA and 50 μl of 10 mg/mllysozyme chloride (Nacalai Tesque) in water was added thereto. Themixture was reacted at 20° C. for 1 hour. After reaction, 4 ml of amixture containing 150 mM NaCl, 1 mM EDTA and 20 mM tris-HCl (pH 8.0),50 μl of 20 mg/ml proteinase K (Takara Shuzo) and 250 μl of a 10%aqueous solution of sodium lauryl sulfate were added to the reactionmixture. The mixture was incubated at 37° C. for 1 hour. After reaction,the reaction was subjected to phenol-chloroform extraction and ethanolprecipitation, air-dried and then dissolved in 100 μl of TE buffer toobtain a genomic DNA solution.

(2) Cloning of Middle Portion of RNase HII Gene

Oligonucleotides RN-F1 and RN-R0 represented by SEQ ID NOS:38 and 39were synthesized on the basis of portions conserved among amino acidsequences of various thermostable RNase HIIs.

A PCR was carried out in a volume of 100 μl using 5 μl of theThermococcus litoralis genomic DNA solution as prepared in Example 8-(1)as a template, and 100 pmol each of RN-F1 and RN-R0 as primers. TaKaRaTaq (Takara Shuzo) was used as a DNA polymerase for the PCR according tothe attached protocol. The PCR was carried out as follows: 50 cycles of94° C. for 30 seconds, 45° C. for 30 seconds and 72° C. for 1 minute.After reaction, Microcon-100 (Takara Shuzo) was used to remove primersfrom the reaction mixture and to concentrate the reaction mixture.

(3) Cloning of Upstream and Downstream Portions of RNase HII Gene

The nucleotide sequence of the inserted fragment of about 0.5 kb,TliF1R0, obtained in Example 8-(2) was determined. A specificoligonucleotide TliRN-1 (SEQ ID NO:40) for cloning the upstream portionand a specific oligonucleotide TliRN-2 (SEQ ID NO:41) for cloning thedownstream portion were synthesized on the basis of the determinednucleotide sequence. In addition, 48 primers as shown in Table 1 weresynthesized. The tag sequence in Table 1 is shown in SEQ ID NO:60.

TABLE 1 5′-tag sequence-NN-SSSSSSS-3′ (N: mixture of G, A, T and C; Srepresents the nucleotide sequence below) Nucleotide No. sequence 1ggagcag 2 ggcaaag 3 ggcaacg 4 ggcacag 5 ggcattg 6 ggccaag 7 ggccttg 8ggctaag 9 ggctacg 10 ggctcag 11 ggctttg 12 gggacag 13 gggcaag 14 gggcttg15 gggtacg 16 ggtaacg 17 ggtacgg 18 ggtagcg 19 gtaacgg 20 gtaagcg 21gtacacg 22 gtagacg 23 gtagcgg 24 gtcaacg 25 gcaccag 26 gcagacg 27gcagcag 28 gcatggg 29 gccaaag 30 gccacag 31 gccattg 32 gcccaag 33gcccttg 34 gcctacg 35 gcctcag 36 gcctttg 37 gcgcaag 38 gcgcttg 39gcggacg 40 gcgtaag 41 gctacgg 42 gctcacg 43 gctccag 44 gcttgcg 45gcttggg 46 ggacacg 47 ggaccag 48 ggagacg

PCRs were carried out in reaction mixtures containing 1 μl of theThermococcus litoralis genomic DNA solution prepared in Example 8-(1) asa template, a combination of 20 pmol of TliRN-1 or 20 pmol of TliRN-2and 20 pmol of one of the 48 primers listed in Table 1, 20 mMtris-acetate (pH 8.5), 50 mM potassium acetate, 3 mM magnesium acetate,0.01% BSA, 30 μM each of dNTPs and 2.5 units of TaKaRa Ex Taq DNApolymerase (Takara Shuzo). PCRs were carried out as follows: incubationat 94° C. for 3 minutes; and 40 cycles of 98° C. for 10 seconds, 50° C.for 10 seconds and 72° C. for 40 seconds. A portion of each PCR productwas subjected to agarose gel electrophoresis. Microcon-100 (TakaraShuzo) was used to remove primers from reaction mixtures that resultedin single bands and to concentrate the reaction mixtures. Theconcentrates were subjected to direct sequencing to screen for fragmentscontaining the upstream or downstream portions of the RNase HII. As aresult, it was shown that an about 450-bp PCR-amplified fragment TliN7contained the upstream portion of the RNase HII gene and an about 600-bpPCR-amplified fragment TliC25 and an about 400-bp PCR-amplified fragmentTliC26 contained the downstream portion of the RNase HII gene,respectively.

(4) Cloning of Entire RNase HII Gene

The nucleotide sequence of a gene containing Tli RNase HII is shown inSEQ ID NO:42. The amino acid sequence of RNase HII deduced from thenucleotide sequence is shown in SEQ ID NO:43. Primers TliNde (SEQ IDNO:44) and TliBam (SEQ ID NO:45) were synthesized on the basis of thenucleotide sequence.

A PCR was carried out in a volume of 100 μl using 1 μl of theThermococcus litoralis genomic DNA solution as prepared in Example 8-(1)as a template, and 20 pmol each of TliNde and TliBam as primers. Ex TaqDNA polymerase (Takara Shuzo) was used as a DNA polymerase for the PCRaccording to the attached protocol. The PCR was carried out as follows:40 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for1 minute. An amplified DNA fragment of about 0.7 kb was digested withNdeI and BamHI (both from Takara Shuzo). Then, plasmids pTLI223Nd andpTLI204 were constructed by incorporating the resulting DNA fragmentbetween NdeI and BamHI sites in a plasmid vector pTV119Nd (a plasmid inwhich the NcoI site in pTV119N is converted into a NdeI site) or pET3a(Novagen), respectively.

(5) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequences of the DNA fragments inserted into pTLI223Ndand pTLI204 obtained in Example 8-(4) were determined according to adideoxy method.

Analyses of the determined nucleotide sequences revealed the existenceof open reading frames each presumably encoding RNase HII. Thenucleotide sequence of the open reading frame in pTLI204 is shown in SEQID NO:46. The amino acid sequence of RNase HII deduced from thenucleotide sequence is shown in SEQ ID NO:47. “T” at position 484 in thenucleotide sequence of the open reading frame in pTLI204 was replaced by“C” in the nucleotide sequence of the open reading frame in pTLI223Nd.In the amino acid sequence, phenylalanine at position 162 was replacedby leucine.

Escherichia coli HMS174(DE3) transformed with the plasmid pTLI204 isdesignated and indicated as Escherichia coli HMS174(DE3)/pTLI204, anddeposited on Feb. 22, 2001 (date of original deposit) at InternationalPatent organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki 305-8566, Japan under accession number FERMBP-7693.

(6) Expression of Thermococcus litoralis RNase HII Gene

Escherichia coli JM109 transformed with pTLI223Nd was inoculated into 10ml of LB medium containing 100 μg/ml of ampicillin and 1 mM IPTG andcultured with shaking at 37° C. overnight. After cultivation, cellscollected by centrifugation were suspended in 196 μl of Buffer A andsonicated. A supernatant obtained by centrifuging the sonicatedsuspension at 12,000 rpm for 10 minutes was heated at 70° C. for 10minutes and then centrifuged again at 12,000 rpm for 10 minutes tocollect a supernatant as a heated supernatant. Similarly, Escherichiacoli HMS174(DE3) transformed with pTLI204 was inoculated into 10 ml ofLB medium containing 100 μg/ml of ampicillin and cultured with shakingat 37° C. overnight. After cultivation, cells collected bycentrifugation were processed according to the procedure as describedabove to obtain a heated supernatant.

The enzymatic activities were measured for the heated supernatants asdescribed in Example 3-(5). As a result, RNase H activities wereobserved for both transformants. Thus, the activity of the polypeptideof the present invention was confirmed in spite of substitution in thenucleotide sequence or the amino acid sequence.

(7) Preparation of Purified RNase HII Preparation

Escherichia coli JM109 transformed with pTLI223Nd obtained in Example8-(4) was inoculated into 2 L of LB medium containing 100 μg/ml ofampicillin and cultured with shaking at 37° C. for 16 hours. Aftercultivation, cells collected by centrifugation were suspended in 38.7 mlof a sonication buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12,000 rpm for 10 minutes washeated at 70° C. for 15 minutes. It was then centrifuged again at 12,000rpm for 20 minutes to collect a supernatant. Thus, 37.2 ml of a heatedsupernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

The sample was subjected to RESOURSE S column (Amersham PharmaciaBiotech) equilibrated with Buffer A and eluted with a linear gradient of0 to 500 mM NaCl using FPLC system (Amersham Pharmacia Biotech). Afraction containing RNase HII eluted with about 220 mM NaCl wasobtained.

A mixture containing 3 ml of the RNase HII fraction and Buffer A addedto make the NaCl concentration to 50 mM was subjected to HiTrap-heparincolumn (Amersham Pharmacia Biotech) equilibrated with Buffer Acontaining 50 mM NaCl and eluted with a linear gradient of 50 to 550 mMNaCl using FPLC system. As a result, a fraction containing RNase HIIeluted with about 320 mM NaCl was obtained.

6 ml of the RNase HII fraction was concentrated by ultrafiltration usingCentricon-10 (Millipore). About 198 μl of the concentrate was subjectedto Superose 6 gel filtration column (Amersham Pharmacia Biotech)equilibrated with 50 mM tris-HCl (pH 8.0) containing 100 mM NaCl and 0.1mM EDTA and eluted with the same buffer. As a result, RNase HII waseluted at a position corresponding to a molecular weight of 26.5kilodalton. This molecular weight corresponds to that of the RNase HIIin the monomeric form. The thus eluted RNase HII was used as Tli RNaseHII preparation.

The enzymatic activity of the thus obtained Tli RNase HII preparationwas measured as described in Example 3-(5). As a result, an RNase Hactivity was observed for the Tli RNase HII preparation.

Example 9 Cloning of Thermococcus celer RNase HII Gene

(1) Preparation of Genomic DNA from Thermococcus celer

Thermococcus celer (purchased from Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH; DSM2476) cells were collected from 11 ml ofculture. A genomic DNA solution was obtained as described in Example8-(1).

(2) Cloning of Middle Portion of RNase HII Gene

Oligonucleotides RN-F1 and RN-R0 represented by SEQ ID NOS:48 and 49were synthesized on the basis of portions conserved among amino acidsequences of various thermostable RNase HIIs.

A PCR was carried out in a volume of 100 μl using 5 μl of theThermococcus celer genomic DNA solution as prepared in Example 9-(1) asa template, and 100 pmol each of RN-F1 and RN-R0 as primers. TaKaRa Taq(Takara Shuzo) was used as a DNA polymerase for the PCR according to theattached protocol. The PCR was carried out as follows: 50 cycles of 94°C. for 30 seconds, 45° C. for 30 seconds and 72° C. for 1 minute. Afterreaction, an about 500-bp DNA fragment obtained by amplification wasblunt-ended using T4 DNA polymerase (Takara Shuzo) and then subjected toagarose gel electrophoresis to recover an amplified DNA fragment ofabout 500 bp. The about 500-bp DNA fragment was ligated with pUC119(Takara Shuzo) digested with SmaI (Takara Shuzo) using T4 DNA ligase(Takara Shuzo). The ligation mixture was used to transform Escherichiacoli JM109.

The resulting transformants were cultured to obtain a plasmid pTceF1R0into which the about 500-bp DNA fragment was inserted.

(3) Cloning of Upstream and Downstream Portions of RNase HII Gene

The nucleotide sequence of the plasmid pTceF1R0 obtained in Example9-(2) was determined. A specific oligonucleotide TceRN-1 (SEQ ID NO:50)for cloning the upstream portion and a specific oligonucleotide TceRN-2(SEQ ID NO:51) for cloning the downstream portion were synthesized onthe basis of the determined nucleotide sequence.

PCRs were carried out in reaction mixtures containing 1 μl of theThermococcus celer genomic DNA solution prepared in Example 9-(1) as atemplate, a combination of 20 pmol of TliRN-1 or 20 pmol of TliRN-2 and20 pmol of one of the 48 primers (listed in Table 1, Example 8), 20 mMtris-acetate (pH 8.5), 50 mM potassium acetate, 3 mM magnesium acetate,0.01% BSA, 30 μM each of dNTPs and 2.5 units of TaKaRa Ex Taq DNApolymerase (Takara Shuzo). PCRs were carried out as follows: incubationat 94° C. for 3 minutes; and 40 cycles of 98° C. for 10 seconds, 50° C.for 10 seconds and 72° C. for 40 seconds. Microcon-100 (Takara Shuzo)was used to remove primers from reaction mixtures that resulted insingle bands for the PCR products and to concentrate the reactionmixtures. The concentrates were subjected to direct sequencing to screenfor fragments containing the upstream or downstream portion of the RNaseHII. As a result, it was shown that an about 450-bp PCR-amplifiedfragment TceN24 contained the upstream portion of the RNase HII gene andan about 400-bp PCR-amplified fragment TceC29 contained the downstreamportion of the RNase HII gene, respectively.

(4) Cloning of Entire RNase HII Gene

The nucleotide sequence of a gene containing Tce RNase HII is shown inSEQ ID NO:52. The amino acid sequence of RNase HII deduced from thenucleotide sequence is shown in SEQ ID NO:53. Primers TceNde (SEQ IDNO:54) and TceBam (SEQ ID NO:55) were synthesized on the basis of thenucleotide sequence.

A PCR was carried out in a volume of 100 μl using 1 μl of theThermococcus celer genomic DNA solution as prepared in Example 9-(1) asa template, and 20 pmol each of TceNde and TceBam as primers. PyrobestDNA polymerase (Takara Shuzo) was used as a DNA polymerase for the PCRaccording to the attached protocol. The PCR was carried out as follows:40 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for1 minute. An amplified DNA fragment of about 0.7 kb was digested withNdeI and BamHI (both from Takara Shuzo). Then, plasmids pTCE265Nd andpTCE207 were constructed by incorporating the resulting DNA fragmentbetween NdeI and BamHI sites in a plasmid vector pTV119Nd (a plasmid inwhich the NcoI site in pTV119N is converted into a NdeI site) or pET3a(Novagen), respectively.

(5) Determination of Nucleotide Sequence of DNA Fragment ContainingRNase HII Gene

The nucleotide sequences of the DNA fragments inserted into pTCE265Ndand pTCE207 obtained in Example 9-(4) were determined according to adideoxy method.

Analyses of the determined nucleotide sequences revealed the existenceof open reading frames each presumably encoding RNase HII. Thenucleotide sequence of the open reading frame in pTCE207 is shown in SEQID NO:56. The amino acid sequence of RNase HII deduced from thenucleotide sequence is shown in SEQ ID NO:57. “A” at position 14 in thenucleotide sequence of the open reading frame in pTCE207 was replaced by“G” in the nucleotide sequence of the open reading frame in pTCE265Ndand nucleotides at positions 693 to 696 were deleted. As a result,glutamic acid at position 5 was replaced by glycine and phenylalanine atposition 231 was missing in the amino acid sequence.

Escherichia coli HMS174(DE3) transformed with the plasmid pTCE207 isdesignated and indicated as Escherichia coli HMS174(DE3)/pTCE207, anddeposited on Feb. 22, 2001 (date of original deposit) at InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki 305-8566, Japan under accession number FERMBP-7694.

(6) Expression of Thermococcus celer RNase HII Gene

Escherichia coli JM109 transformed with pTCE265Nd was inoculated into 10ml of LB medium containing 100 μg/ml of ampicillin and 1 mM IPTG andcultured with shaking at 37° C. overnight. After cultivation, cellscollected by centrifugation were suspended in 203 μl of Buffer A andsonicated. A supernatant obtained by centrifuging the sonicatedsuspension at 12,000 rpm for 10 minutes was heated at 70° C. for 10minutes and then centrifuged again at 12,000 rpm for 10 minutes tocollect a supernatant as a heated supernatant. Similarly, Escherichiacoli HMS174(DE3) transformed with pTCE207 was inoculated into 10 ml ofLB medium containing 100 μg/ml of ampicillin and cultured with shakingat 37° C. overnight. After cultivation, cells collected bycentrifugation were processed according to the procedure as describedabove to obtain a heated supernatant.

The enzymatic activities were measured for the heated supernatants asdescribed in Example 3-(5). As a result, RNase H activities wereobserved for both transformants. Thus, the activity of the polypeptideof the present invention was confirmed in spite of substitution anddeletion in the nucleotide sequence or the amino acid sequence.

(7) Preparation of Purified RNase HII Preparation

Escherichia coli JM109 transformed with pTCE265Nd obtained in Example9-(4) was inoculated into 2 L of LB medium containing 100 μg/ml ofampicillin and cultured with shaking at 37° C. for 16 hours. Aftercultivation, cells collected by centrifugation were suspended in 39 mlof a sonication buffer [50 mM tris-HCl (pH 8.0), 1 mM EDTA, 2 mMphenylmethanesulfonyl fluoride] and sonicated. A supernatant obtained bycentrifuging the sonicated suspension at 12,000 rpm for 10 minutes washeated at 70° C. for 15 minutes. It was then centrifuged again at 12,000rpm for 20 minutes to collect a supernatant. Thus, 37.5 ml of a heatedsupernatant was obtained.

The heated supernatant was subjected to RESOURSE Q column (AmershamPharmacia Biotech) equilibrated with Buffer A [50 mM tris-HCl (pH 8.0),1 mM EDTA] and chromatographed using FPLC system (Amersham PharmaciaBiotech). As a result, RNase HII flowed through the RESOURSE Q column.

The sample was subjected to RESOURSE S column (Amersham PharmaciaBiotech) equilibrated with Buffer A and eluted with a linear gradient of0 to 500 mM NaCl using FPLC system (Amersham Pharmacia Biotech). As aresult, a fraction containing RNase HII eluted with about 220 mM NaClwas obtained.

A mixture containing 3 ml of the RNase HII fraction and Buffer A addedto make the NaCl concentration to 50 mM was subjected to HiTrap-heparincolumn (Amersham Pharmacia Biotech) equilibrated with Buffer Acontaining 50 mM NaCl and eluted with a linear gradient of 50 to 550 mMNaCl using FPLC system. As a result, a fraction containing RNase HIIeluted with about 415 mM NaCl was obtained. 6 ml of the RNase HIIfraction was concentrated by ultrafiltration using Centricon-10. About178 μl of the concentrate was subjected to Superose 6 gel filtrationcolumn (Amersham Pharmacia Biotech) equilibrated with 50 mM tris-HCl (pH8.0) containing 100 mM NaCl and 0.1 mM EDTA and eluted with the samebuffer. As a result, RNase HII was eluted at a position corresponding toa molecular weight of 29.5 kilodalton. This molecular weight correspondsto that of the RNase HII in the monomeric form. The thus eluted RNaseHII was used as Tce RNase HII preparation.

The enzymatic activity of the thus obtained Tee RNase HII preparationwas measured as described in Example 3-(5). As a result, an RNase Hactivity was observed for the Tce RNase HII preparation.

Example 10

Homology searches were conducted for the amino acid sequences andnucleotide sequences from Bacillus caldotenax (hereinafter referred toas BCA), Pyrococcus furiosus (PFU), Thermotoga maritima (TMA),Archaeoglobus fulgidus (AFU), Thermococcus litoralis (TLI), Thermococcusceler (TCE) and Pyrococcus horikoshii (PHO) obtained in Examples 2 to 9.Calculation was conducted using Maximum Matching in DNASIS-Mac (TakaraShuzo) or a computer algorithm FASTA (version 3.0; Pearson, W. R. etal., Pro. Natl. Acad. Sci., 85:2444-2448, 1988) as a search program.

Homologies of the PFU amino acid sequence to the PHO, AFU, TLI and TCEamino acid sequences as determined using DNASIS were 69%, 45%, 65% and58%, respectively. Homologies of the PFU nucleotide sequence to the PHO,AFU, TLI and TCE nucleotide sequences as determined using DNASIS were68%, 60%, 65% and 61%, respectively.

Gene database searches were conducted for PHO, AFU and TMA using thecomputer algorithm FASTA. For the PHO amino acid sequence, the highesthomology to an amino acid sequence presumed to be of a ribonuclease was70% and the lowest was 20%. The homology of PHO to AFU was 50% and thehomology of PHO to TMA was 35%. For the AFU amino acid sequence, thehighest homology was 50% and the lowest was 25%. The amino acid sequencehomology of AFU to TMA was 32%. For the TMA amino acid sequence, thehighest homology was 52% and the lowest was 22%.

Gene database searches were conducted for BCA, PFU, TCE and TLI usingthe computer algorithm BLAST. For the BCA ribonuclease HII, BCAribonuclease HIII, PFU, TCE and TLI amino acid sequences, the highesthomologies to amino acid sequences presumed to be of ribonucleases were43%, 46%, 68%, 70% and 64%, respectively.

In addition, the amino acid sequence homology of PHO to AFU asdetermined using DNASIS was also 50%. The value determined using thecomputer algorithm FASTA was 50% as described above. Thus, it was shownthat there was no significant difference between homology valuesobtained using DNASIS and the computer algorithm FASTA.

Example 11 Modes of Action and Properties of Various RNase Hs

(1) Mode of Action of Bca RNase HIII

A substrate was prepared as follows in order to compare the fashions ofcleavage of Bca RNase HIII and E. coli RNase HI. A PCR was carried outusing a heat extract of Escherichia coli 0157 as a template, a 5′FITC-labeled chimeric primer VT2-R280N3-17 (SEQ ID NO:61) in which threenucleotides from the 3′ end are RNAs, and a DNA primer VT2-F110 (SEQ IDNO:62) to obtain a DNA fragment having three RNAs in one of the twostrands. Microcon-100 (Millipore) was used to remove primers from thePCR product to obtain a substrate for cleavage with RNase H.

39.2 μl of a reaction buffer (20 mM Hepes-KOH (pH 7.8), 1% dimethylsulfoxide, 0.01% bovine serum albumin, 100 mM potassium acetate, 4 mMmagnesium acetate, 0.002% propyrenediamine) containing 0.3 pmol of thesubstrate was prepared. 0.8 μl of E. coli RNase HI 30 U/μl (TakaraShuzo) or a 10-fold dilution of the purified preparation of Bca RNaseHIII obtained in Example 3-(5) with Buffer A was added thereto. Themixtures were reacted at 55° C. for 5 or 10 minutes. After reaction, 2μl each of the reaction mixtures was subjected to electrophoresis ondenaturing 10% acrylamide gel to determined the sizes of cleaved DNAfragments.

In case of E. coli RNase HI, signals appeared at positions of 18 basesand 19 bases. The signal at the position of 19 bases decreased overtime. Background at a position of 20 bases due to incomplete removal ofprimers also decreased over time. In case of Bca RNase HIII, a signalappeared at a position of 19 bases. No decrease in primer background wasobserved.

Based on the above, E. coli RNase HI cleaves at two sites between RNAs.In addition, it was considered that E. coli RNase HI had an activity ofcleaving on the 5′ side of a 3′ RNA and further cleaving on the 5′ sideof a 3′ RNA which was not attached to a DNA. On the other hand, it wasconsidered that Bca RNase HIII only cleaved on the 5′ side of a 3′ RNAand did not cleave if DNA was not attached on the 3′ side of an RNA.Thus, it was considered that the cleavage site selectivity of Bca RNaseHIII was higher than that of B. coli RNase HI.

(2) Modes of Action of Pfu RNase HII, Pho RNase HII and Afu RNase HII

Substrates were prepared as follows in order to analyze the fashions ofcleavage of Pfu (Pyrococcus furiosus) RNase HII, Pho (Pyrococcushorikoshii) RNase HII and Afu (Archaeoglobus fulgidus) RNase HII. PCRswere carried out using a heat extract of Escherichia coli 0157 as atemplate, a 5′ FITC-labeled chimeric primer VT2-IF20N3 (SEQ ID NO:63) inwhich three nucleotides from the 3′ end are RNAS, a 5′ FITC-labeledchimeric primer VT2-IF19N2 (SEQ ID NO:64) in which two nucleotides fromthe 3′ end are RNAs or a 5′ FITC-labeled chimeric primer VT2-IF18N1 (SEQID NO:65) in which one nucleotide at the 3′ end is RNA, and a DNA primerVT2IR20 (SEQ ID NO:66) to obtain DNA fragments VFN3, VFN2 and VFN1 whichhad three, two and one RNA(s) in one of the two strands, respectively.Microcon-100 was used to remove primers from the PCR products to obtainsubstrates for cleavage with RNase H.

39 μl of a reaction buffer (20 mM Hepes-KOH (pH 7.8), 1% dimethylsulfoxide, 0.01% bovine serum albumin, 100 mM potassium acetate, 4 mMmagnesium acetate, 0.002% propyrenediamine) containing 0.3 pmol of oneof the substrates was prepared. 1 μl of one of the purified preparationsof the respective RNase Hs at a concentration of 37.2 U/μl was addedthereto. The mixtures were reacted at 55° C. for 5 or 10 minutes. Afterreaction, 2 μl each of the reaction mixtures was subjected toelectrophoresis on denaturing 10% acrylamide gel to determined the sizesof cleaved DNA fragments.

For each of Pfu RNase HII, Pho RNase HII and Afu RNase HII, a signal wasobserved at a position of 19 bases using VFN3 as a substrate. For eachof Pfu RNase HII, Pho RNase HII and Afu RNase HII, a signal was observedat a position of 18 bases using VFN2 as a substrate. For each of PfuRNase HII and Pho RNase HII, a signal was observed at a position of 17bases using VFN1 as a substrate.

Based on the above, Pfu RNase HII, Pho RNase HII and Afu RNase HIIcleaved on the 5′ side of a 3′ RNA. Pfu RNase HII and Pho RNase HIIcleaved on the 5′ side of an RNA even if the number of RNA was one.There has been no report on an RNase H that cleaves even if the numberof RNA is one. Since the intensities of the signals upon cleavage weresimilar regardless of the number of RNAs, it was shown that there was nodifference in cleavage efficiency depending on the number of RNAs. Inaddition, since decrease in an appeared signal over time or appearanceof a shorter signal was not observed, it was shown that no cleavagetakes place if DNA was not attached on the 3′ side of an RNA.

(3) Ion Requirements of Bca RNase HII and Tma RNase HII

According to the method for measuring an RNase H activity as describedin Example 1, Bca RNase HII and Tma RNase HII required Mn²⁺ and did notexhibit an activity in the presence of Mg²⁺ at all. Comparison ofcleavage in the presence of Mg²⁺ or Mn²⁺ was carried out using VFN3 asdescribed in Example 11-(2) as a substrate for them.

39.2 μl of a reaction buffer (20 mM Hepes-KOH (pH 7.8), 1% dimethylsulfoxide, 0.01% bovine serum albumin, 100 mM potassium acetate, 4 mMmagnesium acetate, 0.002% propyrenediamine) containing 0.3 pmol of thesubstrate and 39.2 μl of a Mn+ reaction buffer (20 mM Hepes-KOH (pH7.8), 1% dimethyl sulfoxide, 0.01% bovine serum albumin, 100 mMpotassium acetate, 10 mM manganese chloride, 0.002% propyrenediamine)containing 0.3 pmol of the substrate were prepared. 0.8 μl of E. coliRNase HI (30 U/μl; Takara Shuzo), a 10-fold dilution of the purifiedpreparation of Bca RNase HII (obtained in Example 2-(6)) with Buffer Aor a 25-fold dilution of the crude cell extract of Tma RNase HII(obtained in Example 5-(3)) with Buffer A was added thereto. Themixtures were reacted at 55° C. for 5 or 10 minutes. After reaction, 2μl each of the reaction mixtures was subjected to electrophoresis ondenaturing 10% acrylamide gel to determined the sizes of cleaved DNAfragments.

When Bca RNase HII or Tma RNase HII was used in the presence of Mg²⁺ orMn²⁺, a signal appeared at a position of 19 bases in each case. Thelevels of the signals were equivalent each other.

Bca RNase HII and Tma RNase HII did not exhibit an activity in thepresence of Mg²⁺ at all according to the method for measuring an RNase Hactivity as described in Example 1. However, they exhibited cleavageactivities in the presence of Mg²⁺ which were equivalent to thoseobserved in the presence of Mn²⁺ as described above.

Base on the above, it was shown that the enzymes may not require Mn²⁺for cleavage depending on the form of a substrate, it may be possible tosubstitute Mg²⁺ for Mn²⁺, and a reaction in the same reaction mixturefor an enzyme that requires Mn²⁺ or Mg²⁺ may be possible.

(4) Examination of Thermostability of RNase H

Thermostabilities were examined using Escherichia coli transformed withthe following: pRHB11 and pBCA3Nd obtained in Examples 2-(5) and 3-(4)for Bacillus caldotenax RNase H; pPFU220 obtained in Example 4-(2) forPyrococcus furiosus RNase H; pTM-RNH obtained in Example 5-(2) forThermotoga maritima RNase H; pPHO238 obtained in Example 6-(2) forPyrococcus horikoshii RNase H; pAFU204 obtained in Example 7-(2) forArchaeoglobus fulgidus RNase H; pTLI204 and pTLI223Nd obtained Example8-(4) for Thermococcus litoralis RNase H; and pTCE207 and pTCE265Ndobtained in Example 9-(4) for Thermococcus celer RNase H. The E. colistrains were cultured, crude enzyme extracts prepared from the cultureswere heated at 60° C. for 15 minutes, and the RNase H activities weredetermined according to the method as described in Example 1. As aresult, RNase H activities were observed for the RNase Hs from all ofthe strains.

INDUSTRIAL APPLICABILITY

The present invention provides a polypeptide having an RNase H activitywhich is highly valuable for genetic engineering, a gene encoding saidpolypeptide and a method for producing said polypeptide by geneticengineering. Since the RNase H of the present invention is thermostable,the present invention provide a method for producing an RNase H which isindustrially advantageous.

It is now possible to use the RNase H of the present invention forvarious uses according to the present invention.

SEQUENCE LISTING FREE TEXT

SEQ ID NO:1: PCR primer BsuII-3 for cloning a gene encoding apolypeptide having a RNase HII activity from Bacillus caldotenax.

SEQ ID NO:2: PCR primer BsuII-6 for cloning a gene encoding apolypeptide having a RNase HII activity from Bacillus caldotenax.

SEQ ID NO:3: PCR primer RNII-S1 for cloning a gene encoding apolypeptide having a RNase HII activity from Bacillus caldotenax.

SEQ ID NO:4: PCR primer RNII-S2 for cloning a gene encoding apolypeptide having a RNase HII activity from Bacillus caldotenax.

SEQ ID NO:5: PCR primer RNII-S5 for cloning a gene encoding apolypeptide having a RNase HII activity from Bacillus caldotenax.

SEQ ID NO:6: PCR primer RNII-S6 for cloning a gene encoding apolypeptide having a RNase HII activity from Bacillus caldotenax.

SEQ ID NO:7: PCR primer RNII-Nde for cloning a gene encoding apolypeptide having a RNase HII activity from Bacillus caldotenax.

SEQ ID NO:10: PCR primer BsuIII-1 for cloning a gene encoding apolypeptide having a RNase HIII activity from Bacillus caldotenax.

SEQ ID NO:11: PCR primer BsuIII-3 for cloning a gene encoding apolypeptide having a RNase HIII activity from Bacillus caldotenax.

SEQ ID NO:12: PCR primer BsuIII-6 for cloning a gene encoding apolypeptide having a RNase HIII activity from Bacillus caldotenax.

SEQ ID NO:13: PCR primer BsuIII-8 for cloning a gene encoding apolypeptide having a RNase HIII activity from Bacillus caldotenax.

SEQ ID NO:14: PCR primer RNIII-S3 for cloning a gene encoding apolypeptide having a RNase HIII activity from Bacillus caldotenax.

SEQ ID NO:15: PCR primer BcaRNIII-3 for cloning a gene encoding apolypeptide having a RNase HIII activity from Bacillus caldotenax.

SEQ ID NO:18: PCR primer BcaRNIIINde for amplifying a gene encoding apolypeptide having a RNase HIII activity from Bacillus caldotenax.

SEQ ID NO:20: PCR primer 1650Nde for cloning a gene encoding apolypeptide having a RNase HII activity from Pyrococcus furiosus.

SEQ ID NO:21: PCR primer 1650Bam for cloning a gene encoding apolypeptide having a RNase HII activity from Pyrococcus furiosus.

SEQ ID NO:24: PCR primer 915-F1 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermotoga maritima.

SEQ ID NO:25: PCR primer 915-F2 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermotoga maritima.

SEQ ID NO:26: PCR primer 915-R1 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermotoga maritima.

SEQ ID NO:27: PCR primer 915-R2 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermotoga maritima.

SEQ ID NO:29: PCR primer PhoNde for cloning a gene encoding apolypeptide having a RNase HII activity from Pyrococcus horikoshii.

SEQ ID NO:30: PCR primer PhoBam for cloning a gene encoding apolypeptide having a RNase HII activity from Pyrococcus horikoshii.

SEQ ID NO:34: PCR primer AfuNde for cloning a gene encoding apolypeptide having a RNase HII activity from Archaeoglobus fulgidus.

SEQ ID NO:35: PCR primer AfuBam for cloning a gene encoding apolypeptide having a RNase HII activity from Archaeoglobus fulgidus.

SEQ ID NO:38: PCR primer RN-F1 for cloning a gene encoding a polypeptidehaving a RNase HII activity from Thermococcus litoralis.

SEQ ID NO:39: PCR primer RN-R0 for cloning a gene encoding a polypeptidehaving a RNase HII activity from Thermococcus litoralis.

SEQ ID NO:40: PCR primer TliRN-1 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermococcus litoralis.

SEQ ID NO:41: PCR primer TliRN-2 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermococcus litoralis.

SEQ ID NO:44: PCR primer TliNde for amplifying a gene encoding apolypeptide having a RNase HII activity from Thermococcus litoralis.

SEQ ID NO:45: PCR primer TliBam for amplifying a gene encoding apolypeptide having a RNase HIII activity from Thermococcus litoralis.

SEQ ID NO:48: PCR primer RN-F1 for cloning a gene encoding a polypeptidehaving a RNase HII activity from Thermococcus celer.

SEQ ID NO:49: PCR primer RN-R0 for cloning a gene encoding a polypeptidehaving a RNase HII activity from Thermococcus celer.

SEQ ID NO:50: PCR primer TceRN-1 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermococcus celer.

SEQ ID NO:51: PCR primer TceRN-2 for cloning a gene encoding apolypeptide having a RNase HII activity from Thermococcus celer.

SEQ ID NO:54: PCR primer TceNde for amplifying a gene encoding apolypeptide having a RNase HII activity from Thermococcus celer.

SEQ ID NO:55: PCR primer TceBam for amplifying a gene encoding apolypeptide having a RNase HIII activity from Thermococcus celer.

SEQ ID NO:61: Designed chimeric oligonucleotide primer as VT2-R280N3-17for amplifying a portion of vero tox in 2-encoding sequence fromhemorrhagic Escherichia coli 0-157. “Nucleotides 18 to 20 areribonucleotides-other nucleotides are deoxyribonucleotides”

SEQ ID NO:62: Designed oligonucleotide primer as VT2-F110 for amplifyinga portion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli 0-157.

SEQ ID NO:63: Designed chimeric oligonucleotide primer as VT2-IF20N3 foramplifying a VFN3 from hemorrhagic Escherichia coli 0-157. “Nucleotides17 to 19 are ribonucleotides-other nucleotides are deoxyribonucleotides”

SEQ ID NO:64: Designed chimeric oligonucleotide primer as VT2-IF19N2 foramplifying VFN2 from hemorrhagic Escherichia coli 0-157. “Nucleotides 16to 18 are ribonucleotides-other nucleotides are deoxyribonucleotides”

SEQ ID NO:65: Designed chimeric oligonucleotide primer as VT2-IF18N1 foramplifying a VFN1 from hemorrhagic Escherichia coli 0-157. “Nucleotides15 to 17 are ribonucleotides-other nucleotides are deoxyribonucleotides”

SEQ ID NO:66: Designed oligonucleotide primer as VT21R20 for amplifyinga portion of vero toxin 2-encoding sequence from hemorrhagic Escherichiacoli 0-157.

1. A polypeptide having a thermostable ribonuclease H activity, which isselected from the group consisting of: (a) a polypeptide having theamino acid sequence of SEQ ID NO: 9, 17, 23, 32, 37, 57 or 59; (b) apolypeptide encoded by a nucleic acid that is hybridizable to nucleicacids of SEQ ID NO:8, 16, 22, 31, 36, 56 or 58 or complementary strandsthereof under stringent conditions; and (c) a polypeptide having anamino acid sequence that shares at least 90% homology with the aminoacid sequence of SEQ ID NO:9, 17, 23, 32, 37, 57 or
 59. 2. A nucleicacid encoding a polypeptide having thermostable ribonuclease H activity,which is selected from the group consisting of: (a) a nucleic acidencoding a polypeptide having the amino acid sequence of SEQ ID NO: 9,17, 23, 32, 37, 47, 57 or 59; (b) a nucleic acid having the nucleotidesequence of SEQ ID NO:8, 16, 22, 31, 36, 46, 56 or 58; (c) a nucleicacid that is hybridizable to any one of the nucleic acids of SEQ IDNO:8, 16, 22, 31, 36, 46, 56 or 58 or complementary strands thereofunder stringent conditions; and (d) a nucleic acid having a nucleotidesequence that shares at least 90% homology with the nucleotide sequenceof SEQ ID NO:8, 16, 22, 31, 36, 46, 56 or
 58. 3. A recombinant DNAcomprising the nucleic acid defined by claim
 2. 4. A transformanttransformed with the recombinant DNA defined by claim
 3. 5. A method forproducing a polypeptide having a thermostable ribonuclease H activity,the method comprising: culturing the transformant defined by claim 4;and collecting a polypeptide having a thermostable ribonuclease Hactivity from the culture.
 6. A polypeptide having a thermostableribonuclease H activity, which is obtainable by culturing a transformantinto which any one of the plasmids pRHB11 (FERM BP-7655), pBCA3Nd2 (FERMBP-7653), pPFU220 (FERM BP-7654), pTM-RNH (FERM BP-7652), pPHO238 (FERMBP-7692), pAFU204 (FERM BP-7691) and pTCE207 (FERM BP-7694) istransferred.